DNA molecules encoding Macaca mulatta androgen receptor

ABSTRACT

The present invention discloses the isolation and characterization of cDNA molecules encoding novel androgen receptor (AR) protein from  Macaca mulatta . Also within the scope of the disclosure are recombinant vectors, recombinant host cells, methods of screening for modulators of  Macaca mulatta  AR (rhAR) activity, purified proteins and fusion proteins which comprise all or a portion of the rhAR protein, transgenic mice comprising a transgene encoding the rhAR protein, as well as production of antibodies against AR, or epitopes thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority of U.S. provisional applicationSer. No. 60/289,573, filed May 8, 2001.

FIELD OF THE INVENTION

The present invention relates in part to isolated nucleic acid molecules(polynucleotides) which encode a Macaca mulatta (rhesus monkey) androgenreceptor (rhAR) protein. The present invention also relates torecombinant vectors and recombinant hosts which contain a DNA fragmentencoding rhAR, substantially purified, biologically active forms ofrhAR, including precursor and mature forms of the protein, mutantproteins which retain a biological activity of interest, methodsassociated with identifying compounds which modulate rhAR activity, andnon-human animals which have been subject to intervention to effect rhARactivity.

BACKGROUND OF THE INVENTION

The nuclear receptor superfamily, which includes steroid hormonereceptors, are small chemical ligand-inducible transcription factorswhich have been shown to play roles in controlling development,differentiation and physiological function. Isolation of cDNA clonesencoding nuclear receptors reveals several characteristics. First, theNH₂-terminal regions, or the A/B domain, which vary in length betweenreceptors, are hypervariable with low homology between family members.There are three internal regions of conservation, referred to as domainsC, D and E/F. Region C encodes a cysteine-rich region which is referredto as the DNA binding domain (DBD). Regions D and E/F are within theCOOH-terminal section of the protein. Region D encodes the hinge domainwhich is also referred to as the ligand binding domain (LBD). For areview, see Power et al. (1992, Trends in Pharmaceutical Sciences 13:318–323).

The lipophilic hormones that activate steroid receptors are known to beassociated with human diseases. Therefore, the respective nuclearreceptors have been identified as possible targets for therapeuticintervention. For a review of the mechanism of action of various steroidhormone receptors, see Tsai and O'Malley (1994, Annu. Rev. Biochem. 63:451–486).

Recent work with non-steroid nuclear receptors has also shown thepotential as drug targets for therapeutic intervention. This workreports that peroxisome proliferator activated receptor g (PPARg),identified by a conserved DBD region, promotes adipocyte differentiationupon activation and that thiazolidinediones, a class of antidiabeticdrugs, function through PPARg (Tontonoz et al., 1994, Cell 79:1147–1156; Lehmann et al., 1995, J. Biol. Chem. 270(22): 12953–12956;Teboul et al., 1995, J. Biol. Chem. 270(47): 28183–28187). Thisindicates that PPARg plays a role in glucose homeostasis and lipidmetabolism.

Mangelsdorf et al. (1995, Cell 83: 835–839) provide a review of knownmembers of the nuclear receptor superfamily.

U.S. Pat. No. 5,614,620, issued to Liao and Chang on Mar. 25, 1997,discloses nucleotide sequences encoding human and rat androgen receptor,along with the complete amino acid sequence within the open readingframe of the respective androgen receptor.

EP 0 365 657 B1 issued to French et al. Aug. 4, 1999, discloses arecombinant DNA molecule encoding a human androgen receptor, along withthe amino acid sequences of human androgen receptor protein.

Choong et al. (1998, J. Mol. Evol. 47: 334–342) disclose amino acidsequences for non-human primates such as chimpanzee, baboon, lemur andMacaca fascicularis (see SEQ ID NO:6 for nucleotide sequence, see alsoGen Bank Accession No. U94179 for the nucleotide and amino acid sequenceof Macaca fascicularis androgen receptor).

Abdelgadir et al. (1999, Biology of Reproduction 60:1251–1256) disclosea PCR fragment representing a 5′ portion of the Macaca mulatta codingregion (see also Gen Bank Accession No. AF092930).

It would be advantageous to identify additional genes closely related tothe human androgen receptor gene, such as those possessed by nonhumanprimates used for pharmacological investigation, which encode anandrogen receptor protein. Since the androgen receptor plays animportant role in regulating development, reproduction, and maintenanceof bone and muscle, such genes, and their expressed functional proteins,will be useful in assays to select for compounds which modulate thebiological activity of the androgen receptor, especially as thismodulation pertains to bone formation. The present invention addressesand meets these needs by disclosing isolated nucleic acid moleculeswhich encode a full-length Macaca mullata androgen receptor.

SUMMARY OF THE INVENTION

The present invention relates in part to isolated nucleic acid molecules(polynucleotides) which encode a full length Macaca mulatta androgenreceptor (rhAR), and the use of the expressed rhAR or portion thereof inthe identification of androgen selective compounds active in boneformation. The isolated polynucleotides of the present invention encodea non-human primate member of this nuclear receptor superfamily. The DNAmolecules disclosed herein may be transfected into a host cell of choicewherein the recombinant host cell provides a source for substantiallevels of an expressed functional rhAR. Such a functional nuclearreceptor will provide for an effective target for use in screeningmethodology to identify modulators of the androgen receptor, modulatorswhich may be effective in regulating development, reproduction andmaintenance of bone and muscle.

A preferred embodiment of the present invention is disclosed in FIG.1A–C and SEQ ID NO: 1, an isolated DNA molecule encoding rhAR.Nucleotide 1051 is polymorphic, present as either a ‘A’ nucleotide or a‘G’ nucleotide (see SEQ ID NO:3).

To this end, another preferred embodiment of the present invention is anisolated DNA molecule as shown in FIG. 1A–C and SEQ ID NO:1, exceptnucleotide 1051 is a ‘G’ nucleotide instead of a ‘A’ nucleotide; thisisolated DNA molecule being additionally disclosed as SEQ ID NO:3.

The present invention also relates to isolated nucleic acid fragmentswhich encode mRNA expressing a biologically active rhesus monkeyandrogen receptor which belongs to the nuclear receptor superfamily. Apreferred embodiment relates to isolated nucleic acid fragments of SEQID NOs:1, and 3 which encode mRNA expressing a biologically functionalderivative of rhAR, especially such nucleic acid fragments which encodeall or a portion of the LBD and/or DBD regions of the rhAR open readingframe.

The present invention also relates to recombinant vectors andrecombinant hosts, both prokaryotic and eukaryotic, transfected and/ortransformed to contain the substantially purified nucleic acid moleculesdisclosed throughout this specification.

A preferred aspect of the present invention relates to a substantiallypurified form of the novel nuclear trans-acting receptor protein, arhesus androgen receptor protein, which is disclosed in FIG. 2 (SEQ IDNO:2) as well as allelic variants of the protein disclosed in SEQ IDNO:2. One allelic variant is disclosed herein as SEQ ID NO:4. TheGlu-210 residue of rhAR of SEQ ID NO:2 the parental allele. A singlenucleotide change at nucleotide 1051 from ‘A’ (of SEQ ID NO:1) to ‘G’(of SEQ ID NO:3) results in an amino acid change at residue 210 of therhAR, from the Glu residue of SEQ ID NO:2 to a Gly-210 residue asdisclosed in SEQ ID NO:4 as the allelic variant.

Another preferred aspect of the present invention relates to asubstantially purified, fully processed (including any proteolyticprocessing, glycosylation and/or phosphorylation) mature rhAR proteinobtained from a recombinant host cell containing a DNA expression vectorcomprising a nucleotide sequence as set forth in SEQ ID NOs: 1 and 3, ornucleic acid fragments thereof as described above, such DNA expressionvectors expressing the respective rhAR protein or rhAR precursorprotein. It is especially preferred that the recombinant host cell be aeukaryotic host cell, including but not limited to a mammalian cellline, insect cell line, or yeast.

The present invention also relates to biologically functionalderivatives of rhAR as set forth as SEQ ID NOs:2 and 4, including butnot limited to rhAR mutants and biologically active fragments such asamino acid substitutions, deletions, additions, amino terminaltruncations and carboxy-terminal truncations, such that these fragmentsprovide for proteins or protein fragments of diagnostic, therapeutic orprophylactic use and would be useful for screening for agonists and/orantagonists of rhAR function.

The present invention also relates to a non-human transgenic animalwhich is useful for studying the ability of a variety of compounds toact as modulators of rhAR, or any alternative functional rhAR in vivo byproviding cells for culture, in vitro. In reference to the transgenicanimals of this invention, reference is made to transgenes and genes. Asused herein, a transgene is a genetic construct including a gene. Thetransgene is integrated into one or more chromosomes in the cells in ananimal by methods known in the art. Once integrated, the transgene iscarried in at least one place in the chromosomes of a transgenic animal.Of course, a gene is a nucleotide sequence that encodes a protein, suchas one or a combination of the cDNA clones described herein. The geneand/or transgene may also include genetic regulatory elements and/orstructural elements known in the art. A type of target cell fortransgene introduction is the embryonic stem cell (ES). ES cells can beobtained from pre-implantation embryos cultured in vitro and fused withembryos (Evans et al., 1981, Nature 292:154–156; Bradley et al., 1984,Nature 309:255–258; Gossler et al., 1986, Proc. Natl. Acad. Sci. USA83:9065–9069; and Robertson et al., 1986 Nature 322:445–448). Transgenescan be efficiently introduced into the ES cells by a variety of standardtechniques such as DNA transfection, microinjection, or byretrovirus-mediated transduction. The resultant transformed ES cells canthereafter be combined with blastocysts from a non-human animal. Theintroduced ES cells thereafter colonize the embryo and contribute to thegerm line of the resulting chimeric animal (Jaenisch, 1988, Science 240:1468–1474). It will also be within the purview of the skilled artisan toproduce transgenic or knock-out invertebrate animals (e.g., C. elegans)which express the rhAR transgene in a wild type background as well in C.elegans mutants knocked out for one or both of the rhAR subunits. Theseorganisms will be helpful in further determining the dominant negativeeffect of rhAR as well as selecting from compounds which modulate thiseffect.

The present invention also relates to a non-human transgenic animalwhich is heterozygous for a functional rhAR gene native to that animal.As used herein, functional is used to describe a gene or protein that,when present in a cell or in vitro system, performs normally as if in anative or unaltered condition or environment. The animal of this aspectof the invention is useful for the study of the specific expression oractivity of rhAR in an animal having only one functional copy of thegene. The animal is also useful for studying the ability of a variety ofcompounds to act as modulators of rhAR activity or expression in vivoor, by providing cells for culture, in vitro. It is reiterated that asused herein, a modulator is a compound that causes a change in theexpression or activity of rhAR, or causes a change in the effect of theinteraction of rhAR with its ligand(s), or other protein(s). In anembodiment of this aspect, the animal is used in a method for thepreparation of a further animal which lacks a functional native AR gene.In another embodiment, the animal of this aspect is used in a method toprepare an animal which expresses the non-native rhAR gene in theabsence of the expression of a native AR gene. In particular embodimentsthe non-human animal is a mouse.

In reference to the transgenic animals of this invention, reference ismade to transgenes and genes. As used herein, a transgene is a geneticconstruct including a gene. The transgene is integrated into one or morechromosomes in the cells in an animal by methods known in the art. Onceintegrated, the transgene is carried in at least one place in thechromosomes of a transgenic animal. Of course, a gene is a nucleotidesequence that encodes a protein, such as rhAR. The gene and/or transgenemay also include genetic regulatory elements and/or structural elementsknown in the art.

An aspect of this invention is a method of producing transgenic animalshaving a transgene including the non-native rhAR gene on a native ARnull background. The method includes providing transgenic animals ofthis invention whose cells are heterozygous for a native gene encoding afunctional rhAR protein and an altered native AR gene. These animals arecrossed with transgenic animals of this invention that are hemizygousfor a transgene including a non-native rhAR gene to obtain animals thatare both heterozygous for an altered native AR gene and hemizygous for anon-native rhAR gene. The latter animals are interbred to obtain animalsthat are homozygous or hemizygous for the non-native rhAR and arehomozygous for the altered native AR gene. In particular embodiments,cell lines are produced from any of the animals produced in the steps ofthe method.

The transgenic animals of this invention are also useful in studying thetissue and temporal specific expression patterns of a non-native rhARthroughout the animals. The animals are also useful in determining theability for various forms of wild-type and mutant alleles of anon-native rhAR to rescue the native AR null deficiency. The animals arealso useful for identifying and studying the ability of a variety ofcompounds to act as modulators of the expression or activity of anon-native rhAR in vivo, or by providing cells for culture, for in vitrostudies.

Of particular interest are transgenic mice with rhAR where rhARexpression dominates mouse endogenous AR and can be turned on tissuespecifically.

As used herein, a “targeted gene” or “Knockout” (KO) is a DNA sequenceintroduced into the germline of a non-human animal by way of humanintervention, including but not limited to, the methods describedherein. The targeted genes of the invention include nucleic acidsequences which are designed to specifically alter cognate endogenousalleles. An altered AR gene should not fully encode the same AR asnative to the host animal, and its expression product can be altered toa minor or great degree, or absent altogether. In cases where it isuseful to express a non-native rhAR gene in a transgenic animal in theabsence of a native AR gene we prefer that the altered AR gene induce anull lethal knockout phenotype in the animal. However a more modestlymodified AR gene can also be useful and is within the scope of thepresent invention.

A type of target cell for transgene introduction is the embryonic stemcell (ES). ES cells can be obtained from pre-implantation embryoscultured in vitro and fused with embryos (Evans et al., 1981, Nature292:154–156; Bradley et al., 1984, Nature 309:255–258; Gossler et al.,1986, Proc. Natl. Acad. Sci. USA 83:9065–9069; and Robertson et al.,1986 Nature 322:445–448). Transgenes can be efficiently introduced intothe ES cells by a variety of standard techniques such as DNAtransfection, microinjection, or by retrovirus-mediated transduction.The resultant transformed ES cells can thereafter be combined withblastocysts from a non-human animal. The introduced ES cells thereaftercolonize the embryo and contribute to the germ line of the resultingchimeric animal (Jaenisch, 1988, Science 240: 1468–1474).

The methods for evaluating the targeted recombination events as well asthe resulting knockout mice are readily available and known in the art.Such methods include, but are not limited to DNA (Southern)hybridization to detect the targeted allele, polymerase chain reaction(PCR), polyacrylamide gel electrophoresis (PAGE) and Western blots todetect DNA, RNA and protein.

The present invention also relates to polyclonal and monoclonalantibodies raised in response to rhAR, or a biologically functionalderivative thereof. In particular, antibodies to the A/B domain and thehinge domain, (D domain) are preferred. To this end, the DNA molecules,RNA molecules, recombinant protein and antibodies of the presentinvention may be used to screen and measure levels of rhAR. Therecombinant proteins, DNA molecules, RNA molecules and antibodies lendthemselves to the formulation of kits suitable for the detection andtyping of rhAR.

The present invention also relates assays utilized to identify compoundsthat modulate rhAR activity. One aspect of this portion of the inventionis shown in Example Section 2, an in vitro binding assay using aGST-rhARLBD fusion protein. Other assays are contemplated, including butnot limited to using rhAR cDNA clones and/or expressed proteins inco-transfection assays to measure bioactivity of compounds, as well asmammalian two-hybrid assays to test the effect of compounds on NH₂— andCOOH-terminus interaction of Macaca mulatta AR. Such assays aredescribed infra.

It is an object of the present invention to provide an isolated nucleicacid molecule which encodes a novel form of a nuclear receptor proteinsuch as human rhAR, human nuclear receptor protein fragments of fulllength proteins such as rhAR, and mutants which are derivatives of SEQID NOs:2 and 4. Any such polynucleotide includes but is not necessarilylimited to nucleotide substitutions, deletions, additions,amino-terminal truncations and carboxy-terminal truncations such thatthese mutations encode mRNA which express a protein or protein fragmentof diagnostic, therapeutic or prophylactic use and would be useful forscreening for agonists and/or antagonists for rhAR function.

Another object of this invention is tissue typing using probes orantibodies of this invention. In a particular embodiment, polynucleotideprobes are used to identify tissues expressing rhAR mRNA. In anotherembodiment, probes or antibodies can be used to identify a type oftissue based on rhAR expression or display of rhAR receptors.

It is a further object of the present invention to provide rhAR proteinsor protein fragments encoded by the nucleic acid molecules referred toin the preceding paragraphs, including such rhAR proteins which areexpressed within host cells transfected with a DNA expression vectorwhich contains an rhAR nucleotide sequence as disclosed herein.

It is a further object of the present invention to provide recombinantvectors and recombinant host cells which comprise a nucleic acidsequence encoding rhAR or a biological equivalent thereof.

It is an object of the present invention to provide a substantiallypurified form of rhAR, as set forth in SEQ ID NOs:2 and 4.

It is an object of the present invention to provide for biologicallyfunctional derivatives of rhAR, including but not necessarily limited toamino acid substitutions, deletions, additions, amino terminaltruncations and carboxy-terminal truncations such that these fragmentand/or mutants provide for proteins or protein fragments of diagnostic,therapeutic or prophylactic use.

It is also an object of the present invention to provide for rhAR-basedin-frame fusion constructions, methods of expressing these fusionconstructions and biological equivalents disclosed herein, relatedassays, recombinant cells expressing these constructs, the expressedfusion proteins, and agonistic and/or antagonistic compounds identifiedthrough the use of DNA molecules encoding these rhAR-based fusionproteins. A preferred fusion construct is one which encodes all or aportion of the LBD and/or DBD regions of the rhAR open reading frame. Apreferred fusion protein is one which is expressed from such aconstruct.

It is also an object of the present invention to provide for assays toidentify compounds which modulate rhAR activity.

As used herein, “AR” refers to—androgen receptor—.

As used herein, “rhAR” refers to—Macaca mulatta androgen receptor

As used herein, “DBD” refers to—DNA binding domain—.

As used herein, “LBD” refers to—ligand binding domain—.

As used herein, “SARM” refers to—selective androgen receptor modulator—.

As used herein, the term “mammalian host” refers to any mammal,including a human being.

As used herein, “R1881” refers to methyltrieneolone, also known as17b-hydroxy-17-methylestra-4,9,11-trien-3-one, the preparation of whichis described in Vellux et al., 1963, Compt. Rend. 257: 569 et seq.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A–C shows the nucleotide sequence (SEQ ID NO: 1) which comprisesthe open reading frame encoding the rhAR. Underlined nucleotide 1051(‘A’) is the site of an allelic variant, which may also be representedby a ‘G’ residue (as disclosed in SEQ ID NO:3).

FIG. 2 shows the amino acid sequence (SEQ ID NO: 2) of rhAR. The regionin bold and underlined (from residue 535 to residue 600 of SEQ ID NO:2)is the DNA binding domain (DBD). Residue 210 (Glu residue also in boldand underlined) is the site of an allelic variant which may also berepresented by a Gly residue (as encoded by SEQ ID NO:3 and disclosedherein as SEQ ID NO:4).

FIG. 3A–F shows the coding (SEQ ID NO:1) and anticoding (SEQ ID NO:5)strands which comprises the open reading frame for the rhesus androgenreceptor protein (SEQ ID NO:2). The underlined portion (i.e., from aminoacid residue 535 to amino acid residue 600 of SEQ ID NO:2) representsthe DBD region of expressed rhAR protein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the identification and cloning of genesencoding full length Macaca mulatta androgen receptor (rhAR) and theiruse in the identification of tissue selective androgen compounds,including those active in bone formation, myoanabolism, treatment ofsarcopenia, relief of post-menopausal symptoms, treatment of benignprostatic hyperplasia, treatment of acne, treatment of hirsutism,treatment of male hypogonadism, prevention and treatment of prostatecancer, management of lipids, treatment of atherosclerosis, preventionand treatment of breast cancer. The androgen receptor is a member of thenuclear receptor superfamily. The superfamily is composed of a group ofstructurally related receptors but regulated by chemically distinctligands. The common structure for them is a conserved DNA binding domain(DBD) located in the center of the peptide and a conservedligand-binding domain (LBD) at the C-terminus. Eight out of the ninenon-variant cysteines form two type II zinc fingers which distinguishthem from other DNA-binding proteins.

The present invention relates to isolated nucleic acid molecules(polynucleotides) which encode novel Macaca mulatta (rhesus monkey)androgen receptor (rhAR). The isolated polynucleotides of the presentinvention encode a non-primate member of this nuclear receptorsuperfamily. The DNA molecules disclosed herein may be transfected intoa host cell of choice wherein the recombinant host cell provides asource for substantial levels of an expressed, substantially purified,functional recombinant rhAR, which also forms a portion of the presentinvention. As noted herein, such a functional nuclear receptor willprovide for an effective target for use in screening methodology toidentify modulators of the androgen receptor, modulators which may beeffective in regulating development, reproduction and maintenance ofbone and muscle, treatment of prostate disease, regulation of lipidmetabolism and hippocampal function. It is also known that abnormalfunction of AR can cause prostate cancer. Accumulated information hasalso indicated that androgen deficiency results in various abnormalitiesof bone metabolism, such as increased bone loss. Androgen therapy hasbeen used widely to treat a variety of disorders in both men and women.However, the development of an androgen modulator with desirable effect(i.e., bone promotion) and less side effect (i.e., aggressive behavior,acne) has not been achieved. Recent progress in hormone replacementtherapy has proven the possibility in developing selective androgenreceptor modulators (SARMs). J. of Clinical Endocrinology & Metabolism,84(10): 3459 (1999). Therefore, a compound screening system using AR,such as the rhAR disclosed herein, is needed for safe androgen drugdevelopment.

A preferred embodiment of the present invention is disclosed in FIG.1A–C and SEQ ID NO: 1, an isolated DNA molecule encoding rhAR.Nucleotide 1051 is polymorphic, present as either a ‘A’ nucleotide or a‘G’ nucleotide (see SEQ ID NO:3). This embodiment is shown as follows,with 1051-A being bolded and underlined:

1 CCCAAAAAAT AAAAACAAAC AAAAACAAAA CAAAACAAAA AAAACGAATA (SEQ ID NO: 1)51 AAGAAAAAGG TAATAACTCA GTTCTTATTT GCACCTACTT CCAGTGGACA 101 CTGAATTTGGAAGGTGGAGG ATTCTTGTTT TTTCTTTTAA GATCGGGCAT 151 CTTTTGAATC TACCCCTCAAGTGTTAAGAG ACAGACTGTG AGCCTAGCAG 201 GGCAGATCTT GTCCACCGTG TGTCTTCTTTTGCAGGAGAC TTTGAGGCTG 251 TCAGAGCGCT TTTTGCGTGG TTGCTCCCGC AAGTTTCCTTCTCTGGAGCT 301 TCCCGCAGGT GGGCAGCTAG CTGCAGCGAC TACCGCATCA TCACAGCCTG351 TTGAACTCTT CTGAGCAAGA GAAGGGGAGG CGGGGTAAGG GAAGTAGGTG 401GAAGATTCAG CCAAGCTCAA GGATGGAGGT GCAGTTAGGG CTGGGGACGG 451 TCTACCCTCGGCCGCCGTCC AAGACCTACC GAGGAGCTTT CCAGAATCTG 501 TTCCAGAGCG TGCGCGAAGTGATCCAGAAC CCGGGCCCCA GGCACCCAGA 551 GGCCGCGAGC GCAGCACCTC CCGGCGCCAGTTTGCAGCAG CAGCAGCAGC 601 AGCAGCAAGA AACTAGCCCC CGGCAACAGC AGCAGCAGCAGCAGGGTGAG 651 GATGGTTCTC CCCAAGCCCA TCGTAGAGGC CCCACAGGCT ACCTGGTCCT701 GGATGAGGAA CAGCAGCCTT CACAGCCTCA GTCAGCCCCG GAGTGCCACC 751CCGAGAGAGG TTGCGTCCCA GAGCCTGGAG CCGCCGTGGC CGCCGGCAAG 801 GGGCTGCCGCAGCAGCTGCC AGCACCTCCG GACGAGGATG ACTCAGCTGC 851 CCCATCCACG TTGTCTCTGCTGGGCCCCAC TTTCCCCGGC TTAAGCAGCT 901 GCTCCGCCGA CCTTAAAGAC ATCCTGAGCGAGGCCAGCAC CATGCAACTC 951 CTTCAGCAAC AGCAGCAGGA AGCAGTATCC GAAGGCAGCAGCAGCGGGAG 1001 AGCGAGGGAG GCCTCGGGGG CTCCCACTTC CTCCAAGGAC AATTACTTAG1051 A GGGCACTTC GACCATTTCT GACAGCGCCA AGGAGCTGTG TAAGGCAGTG 1101TCGGTGTCCA TGGGCTTGGG TGTGGAGGCG TTGGAGCATC TGAGTCCAGG 1151 GGAACAGCTTCGGGGGGATT GCATGTACGC CCCAGTTTTG GGAGTTCCAC 1201 CCGCTGTGCG TCCCACTCCGTGTGCCCCAT TGGCCGAATG CAAAGGTTCT 1251 CTGCTAGACG ACAGCGCAGG CAAGAGCACTGAAGATACTG CTGAGTATTC 1301 CCCTTTCAAG GGAGGTTACA CCAAAGGGCT AGAAGGCGAGAGCCTAGGCT 1351 GCTCTGGCAG CGCTGCAGCA GGGAGCTCCG GGACACTTGA ACTGCCGTCC1401 ACCCTGTCTC TCTACAAGTC CGGAGCACTG GACGAGGCAG CTGCGTACCA 1451GAGTCGCGAC TACTACAACT TTCCACTGGC TCTGGCCGGG CCGCCGCCCC 1501 CTCCACCGCCTCCCCATCCC CACGCTCGCA TCAAGCTGGA GAACCCGCTG 1551 GACTATGGCA GCGCCTGGGCGGCTGCGGCG GCGCAGTGCC GCTATGGGGA 1601 CCTGGCGAGC CTGCATGGCG CGGGTGCAGCGGGACCCGGC TCTGGGTCAC 1651 CCTCAGCGGC CGCTTCCTCA TCCTGGCACA CTCTCTTCACAGCCGAAGAA 1701 GGCCAGTTGT ATGGACCGTG TGGTGGTGGG GGCGGCGGCG GTGGCGGCGG1751 CGGCGGCGGC GCAGGCGAGG CGGGAGCTGT AGCCCCCTAC GGCTACACTC 1801GGCCACCTCA GGGGCTGGCG GGCCAGGAAG GCGACTTCAC CGCACCTGAT. 1851 GTGTGGTACCCTGGCGGCAT GGTGAGCAGA GTGCCCTATC CCAGTCCCAC 1901 TTGTGTCAAA AGCGAGATGGGCCCCTGGAT GGATAGCTAC TCCGGACCTT 1951 ACGGGGACAT GCGTTTGGAG ACTGCCAGGGACCATGTTTT GCCAATTGAC 2001 TATTACTTTC CACCCCAGAA GACCTGCCTG ATCTGTGGAGATGAAGCTTC 2051 TGGGTGTCAC TATGGAGCTC TCACATGTGG AAGCTGCAAG GTCTTCTTCA2101 AAAGAGCCGC TGAAGGGAAA CAGAAGTACC TGTGTGCCAG CAGAAATGAT 2151TGCACTATTG ATAAATTCCG AAGGAAAAAT TGTCCATCTT GCCGTCTTCG 2201 GAAATGTTATGAAGCAGGGA TGACTCTGGG AGCCCGGAAG CTGAAGAAAC 2251 TTGGTAATCT GAAACTACAGGAGGAAGGAG AGGCTTCCAG CACCACCAGC 2301 CCCACTGAGG AGACAGCCCA GAAGCTGACAGTGTCACACA TTGAAGGCTA 2351 TGAATGTCAG CCCATCTTTC TGAATGTCCT GGAGGCCATTGAGCCAGGTG 2401 TGGTGTGTGC TGGACATGAC AACAACCAGC CCGACTCCTT CGCAGCCTTG2451 CTCTCTAGCC TCAATGAACT GGGAGAGAGA CAGCTTGTAC ATGTGGTCAA 2501GTGGGCCAAG GCCTTGCCTG GCTTCCGCAA CTTACACGTG GACGACCAGA 2551 TGGCTGTCATTCAGTACTCC TGGATGGGGC TCATGGTGTT TGCCATGGGC 2601 TGGCGATCCT TCACCAATGTCAACTCCAGG ATGCTCTACT TTGCCCCTGA 2651 TCTGGTTTTC AATGAGTACC GCATGCACAAATCCCGGATG TACAGCCAGT 2701 GTGTCCGAAT GAGGCACCTC TCTCAAGAGT TTGGATGGCTCCAAATCACC 2751 CCCCAGGAAT TCCTGTGCAT GAAAGCGCTG CTACTCTTCA GCATTATTCC2801 AGTGGATGGG CTGAAAAATC AAAAATTCTT TGATGAACTT CGAATGAACT 2851ACATCAAGGA ACTCGATCGT ATCATTGCAT GCAAAAGAAA AAATCCCACA 2901 TCCTGCTCAAGGCGTTTCTA CCAGCTCACC AAGCTCCTGG ACTCCGTGCA 2951 GCCTATTGCG AGAGAGCTGCATCAGTTCAC TTTTGACCTG CTAATCAAGT 3001 CACACATGGT GAGCGTGGAC TTTCCGGAAATGATGGCAGA GATCATCTCT 3051 GTGCAAGTGC CCAAGATCCT TTCTGGGAAA GTCAAGCCCATCTATTTCCA 3101 CACCCAGTGA AGCATTGGAA ATCCCTATTT CCTCACCCCA GCTCATGCCC3151 CCTTTCAGAT GTCTTCTGCC TGTTA.

As noted above, nucleotide 1051 represents a single nucleotidepolymorphism (SNP). To this end, another preferred embodiment of thepresent invention is an isolated DNA molecule as shown in FIG. 1A–C andSEQ ID NO:1, except nucleotide 1051 is a ‘G’ nucleotide instead of a ‘A’nucleotide, this isolated DNA molecule being additionally disclosed asSEQ ID NO:3, as follows, with 1051-G being bolded and underlined:

1 CCCAAAAAAT AAAAACAAAC AAAAACAAAA CAAAACAAAA AAAACGAATA (SEQ ID NO: 3)51 AAGAAAAAGG TAATAACTCA GTTCTTATTT GCACCTACTT CCAGTGGACA 101 CTGAATTTGGAAGGTGGAGG ATTCTTGTTT TTTCTTTTAA GATCGGGCAT 151 CTTTTGAATC TACCCCTCAAGTGTTAAGAG ACAGACTGTG AGCCTAGCAG 201 GGCAGATCTT GTCCACCGTG TGTCTTCTTTTGCAGGAGAC TTTGAGGCTG 251 TCAGAGCGCT TTTTGCGTGG TTGCTCCCGC AAGTTTCCTTCTCTGGAGCT 301 TCCCGCAGGT GGGCAGCTAG CTGCAGCGAC TACCGCATCA TCACAGCCTG351 TTGAACTCTT CTGAGCAAGA GAAGGGGAGG CGGGGTAAGG GAAGTAGGTG 401GAAGATTCAG CCAAGCTCAA GGATGGAGGT GCAGTTAGGG CTGGGGAGGG 451 TCTACCCTCGCCCGCCGTCC AAGACCTACC GAGGAGCTTT CCAGAATCTG 501 TTCCAGAGCG TGCGCGAAGTGATCCAGAAC CCGGGCCCCA GGCACCCAGA 551 GGCCGCGAGC GCAGCACCTC CCGGCGCCAGTTTGCAGCAG CAGCAGCAGC 601 AGCAGCAAGA AACTAGCCCC CGGCAACAGC AGCAGCAGCAGCAGGGTGAG 651 GATGGTTCTC CCCAAGCCCA TCGTAGAGGC CCCACAGGCT ACCTGGTCCT701 GGATGAGGAA CAGCAGCCTT CACAGCCTCA GTCAGCCCCG GAGTGCCACC 751CCGAGAGAGG TTGCGTCCCA GAGCCTGGAG CCGCCGTGGC CGCCGGCAAG 801 GGGCTGCCGCAGCAGCTGCC AGCACCTCCG GACGAGGATG ACTCAGCTGC 851 CCCATCCACG TTGTCTCTGCTGGGCCCCAC TTTCCCCGGC TTAAGCAGCT 901 GCTCCGCCGA CCTTAAAGAC ATCCTGAGCGAGGCCAGCAC CATGCAACTC 951 CTTCAGCAAC AGCAGCAGGA AGCAGTATCC GAAGGGAGGAGCAGCGGGAG 1001 AGCGAGGGAG GCCTCGGGGG CTCCCACTTC CTCCAAGGAC AATTACTTAG1051 G GGGCACTTC GACCATTTCT GACAGCGCCA AGGAGCTGTG TAAGGCAGTG 1101TCGGTGTCCA TGGGCTTGGG TGTGGAGGCG TTGGAGCATC TGAGTCCAGG 1151 GGAACAGCTTCGGGGGGATT GCATGTACGC CCCAGTTTTG GGAGTTCCAC 1201 CCGCTGTGCG TCCCACTCCGTGTGCCCCAT TGGCCGAATG CAAAGGTTCT 1251 CTGCTAGACG ACAGCGCAGG CAAGAGCACTGAAGATACTG CTGAGTATTC 1301 CCCTTTCAAG GGAGGTTACA CCAAAGGGCT AGAAGGCGAGAGCCTACGCT 1351 GCTCTGGCAG CGCTGCAGCA GGGAGCTCCG GGACACTTGA ACTGCCGTCC1401 ACCCTGTCTC TCTACAAGTC CGGAGCACTG GACGAGGCAG CTGCGTACCA 1451GAGTCGCGAC TACTACAACT TTCCACTGGC TCTGGCCGGG CCGCCGCCCC 1501 CTCCACCGCCTCCCCATCCC CACGCTCGCA TCAAGCTGGA GAACCCGCTG 1551 GACTATGGCA GCGCCTGGGCGGCTGCGGCG GCGCAGTGCC GCTATGGGGA 1601 CCTGGCGAGC CTGCATGGCG CGGGTGCAGCGGGACCCGGC TCTGGGTCAC 1651 CCTCAGCGGC CGCTTCCTCA TCCTGGCACA CTCTCTTCACAGCCGAAGAA 1701 GGCCAGTTGT ATGGACCGTG TGGTGGTGGG GGCGGCGGCG GTGGCGGCGG1751 CGGCGGCGGC GCAGGCGAGG CGGGAGCTGT AGCCCCCTAC GGCTACACTC 1801GGCCACCTCA GGGGCTGGCG GGCCAGGAAG GCGACTTCAC CGCACCTGAT 1851 GTGTGGTACCCTGGCGGCAT GGTGAGCAGA GTGCCCTATC CCAGTCCCAC 1901 TTGTGTCAAA AGCGAGATGGGCCCCTGGAT GGATAGCTAC TCCGGACCTT 1951 ACGGGGACAT GCGTTTGGAG ACTGCCAGGGACCATGTTTT GCCAATTGAC 2001 TATTACTTTC CACCCCAGAA GACCTGCCTG ATCTGTGGAGATGAAGCTTC 2051 TGGGTGTCAC TATGGAGCTC TCACATGTGG AAGCTGCAAG GTCTTCTTCA2101 AAAGAGCCGC TGAAGGGAAA CAGAAGTACC TGTGTGCCAG CAGAAATGAT 2151TGCACTATTG ATAAATTCCG AAGGAAAAAT TGTCCATCTT GCCGTCTTCG 2201 GAAATGTTATGAAGCAGGGA TGACTCTGGG AGCCCGGAAG CTGAAGAAAC 2251 TTGGTAATCT GAAACTACAGGAGGAAGGAG AGGCTTCCAG CACCACCAGC 2301 CCCACTGAGG AGACAGCCCA GAAGCTGACAGTGTCACACA TTGAAGGCTA 2351 TGAATGTCAG CCCATCTTTC TGAATGTCCT GGAGGCCATTGAGCCAGGTG 2401 TGGTGTGTGC TGGACATGAC AACAACCAGC CCGACTCCTT CGCAGCCTTG2451 CTCTCTAGCC TCAATGAACT GGGAGAGAGA CAGCTTGTAC ATGTGGTCAA 2501GTGGGCCAAG GCCTTGCCTG GCTTCCGCAA CTTACACGTG GACGACCAGA 2551 TGGCTGTCATTCAGTACTCC TGGATGGGGC TCATGGTGTT TGCCATGGGC 2601 TGGCGATCCT TCACCAATGTCAACTCCAGG ATGCTCTACT TTGCCCCTGA 2651 TCTGGTTTTC AATGAGTACC GCATGCACAAATCCCGGATG TACAGCCAGT 2701 GTGTCCGAAT GAGGCACCTC TCTCAAGAGT TTGGATGGCTCCAAATCACC 2751 CCCCAGGAAT TCCTGTGCAT GAAAGCGCTG CTACTCTTCA GCATTATTCC2801 AGTGGATGGG CTGAAAAATC AAAAATTCTT TGATGAACTT CGAATGAACT 2851ACATCAAGGA ACTCGATCGT ATCATTGCAT GCAAAAGAAA AAATCCCACA 2901 TCCTGCTCAAGGCGTTTCTA CCAGCTCACC AAGCTCCTGG ACTCCGTGCA 2951 GCCTATTGCG AGAGAGCTGCATCAGTTCAC TTTTGACCTG CTAATCAAGT 3001 CACACATGGT GAGCGTGGAC TTTCCGGAAATGATGGCAGA GATCATCTCT 3051 GTGCAAGTGC CCAAGATCCT TTCTGGGAAA GTCAAGCCCATCTATTTCCA 3101 CACCCAGTGA AGCATTGGAA ATCCCTATTT CCTCACCCCA GCTCATGCCC3151 CCTTTCAGAT GTCTTCTGCC TGTTA.

The above-exemplified isolated DNA molecules, comprise the followingcharacteristics:

(SEQ ID NO: 1)—3175 nuc.: initiating Met (nuc. 423–425) and “TCA” term.codon (nuc. 3106–3108), with a polymorphic site at nucleotide 1051(‘A’), the open reading frame resulting in an expressed protein of 895amino acids, as set forth in SEQ ID NO:2, with amino acid residue 210being a Glu (E) residue.(SEQ ID NO:3)—3175 nuc.: initiating Met (nuc. 423–425) and “TCA” term.codon (nuc.3106–3108), with a polymorphic site at nucleotide 1051 (‘G’),the open reading frame resulting in an expressed protein of 895 aminoacids, as set forth in SEQ ID NO:4, with amino acid residue 210 being aGly (G) residue.

The present invention also relates to isolated nucleic acid fragmentswhich encode mRNA expressing a biologically active rhesus monkeyandrogen receptor which belongs to the nuclear receptor superfamily. Apreferred embodiment relates to isolated nucleic acid fragments of SEQID NOs:1 and 3 which encode mRNA expressing a biologically functionalderivative of rhAR. Any such nucleic acid fragment will encode either aprotein or protein fragment comprising at least an intracellularDNA-binding domain and/or ligand binding domain, domains conservedthroughout the rhAR nuclear receptor family domain which exist in rhAR(SEQ ID NOs: 2 and 4). Any such polynucleotide includes but is notnecessarily limited to nucleotide substitutions (including but notlimited to SNPs, such as single nucleotide substitutions as disclosedherein, as well as deletion and/or insertions which fall within theknown working definition of a SNP), deletions, additions, amino-terminaltruncations and carboxy-terminal truncations such that these mutationsencode mRNA which express a protein or protein fragment of diagnostic,therapeutic or prophylactic use and would be useful for screening foragonists and/or antagonists of rhAR.

The isolated nucleic acid molecule of the present invention may includea deoxyribonucleic acid molecule (DNA), such as genomic DNA andcomplementary DNA (cDNA), which may be single (coding or noncodingstrand) or double stranded, as well as synthetic DNA, such as asynthesized, single stranded polynucleotide. The isolated nucleic acidmolecule of the present invention may also include a ribonucleic acidmolecule (RNA). The preferred template is DNA.

It is known that there is a substantial amount of redundancy in thevarious codons which code for specific amino acids. Therefore, thisinvention is also directed to those DNA sequences encode RNA comprisingalternative codons that code for the eventual translation of theidentical amino acid, as shown below:

-   A=Ala=Alanine: codons GCA, GCC, GCG, GCU-   C=Cys=Cysteine: codons UGC, UGU-   D=Asp=Aspartic acid: codons GAC, GAU-   E=Glu=Glutamic acid: codons GAA, GAG-   F=Phe=Phenylalanine: codons UUC, UUU-   G=Gly=Glycine: codons GGA, GGC, GGG, GGU-   H=His=Histidine: codons CAC, CAU-   I=Ile=Isoleucine: codons AUA, AUC, AUU-   K=Lys=Lysine: codons AAA, AAG-   L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU-   M=Met=Methionine: codon AUG-   N=Asp=Asparagine: codons AAC, AAU-   P=Pro=Proline: codons CCA, CCC, CCG, CCU-   Q=Gln=Glutamine: codons CAA, CAG-   R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU-   S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU-   T=Thr=Threonine: codons ACA, ACC, ACG, ACU-   V=Val=Valine: codons GUA, GUC, GUG, GUU-   W=Trp=Tryptophan: codon UGG-   Y=Tyr=Tyrosine: codons UAC, UAU.    Therefore, the present invention discloses codon redundancy that may    result in differing DNA molecules expressing an identical protein.    For purposes of this specification, a sequence bearing one or more    replaced codons will be defined as a degenerate variation. Also    included within the scope of this invention are mutations either in    the DNA sequence or the translated protein, which do not    substantially alter the ultimate physical properties of the    expressed protein. For example, substitution of valine for leucine,    arginine for lysine, or asparagine for glutamine may not cause a    change in functionality of the polypeptide.

It is known that DNA sequences coding for a peptide may be altered so asto code for a peptide having properties that are different than those ofthe naturally occurring peptide. Methods of altering the DNA sequencesinclude but are not limited to site directed mutagenesis. Examples ofaltered properties include but are not limited to changes in theaffinity of an enzyme for a substrate or a receptor for a ligand.

As used herein, “purified” and “isolated” may be utilizedinterchangeably to stand for the proposition that the nucleic acid,protein, or respective fragment thereof in question has beensubstantially removed from its in vivo environment so that it may bemanipulated by the skilled artisan, such as but not limited tonucleotide sequencing, restriction digestion, site-directed mutagenesis,and subcloning into expression vectors for a nucleic acid fragment aswell as obtaining the protein or protein fragment in pure quantities soas to afford the opportunity to generate polyclonal antibodies,monoclonal antibodies, amino acid sequencing, and peptide digestion.Therefore, the nucleic acids claimed herein may be present in wholecells or in cell lysates or in a partially purified or substantiallypurified form. A nucleic acid is considered substantially purified whenit is purified away from environmental contaminants. Thus, a nucleicacid sequence isolated from cells is considered to be substantiallypurified when purified from cellular components by standard methodswhile a chemically synthesized nucleic acid sequence is considered to besubstantially purified when purified from its chemical precursors.

Any of a variety of procedures may be used to clone rhAR. These methodsinclude, but are not limited to, (1) a RACE PCR cloning technique(Frohman, et al., 1988, Proc. Natl. Acad. Sci. USA 85: 8998–9002). 5′and/or 3′ RACE may be performed to generate a full length cDNA sequence.This strategy involves using gene-specific oligonucleotide primers forPCR amplification of rhAR cDNA. These gene-specific primers are designedthrough identification of an expressed sequence tag (EST) nucleotidesequence which has been identified by searching any number of publiclyavailable nucleic acid and protein databases; (2) direct functionalexpression of the rhAR following the construction of a rhAR-containingcDNA library in an appropriate expression vector system; (3) screening arhAR-containing cDNA library constructed in a bacteriophage or plasmidshuttle vector with a labeled degenerate oligonucleotide probe designedfrom the amino acid sequence of the rhAR protein; (4) screening arhAR-containing cDNA library constructed in a bacteriophage or plasmidshuttle vector with a partial cDNA encoding the rhAR protein. Thispartial cDNA is obtained by the specific PCR amplification of rhAR DNAfragments through the design of degenerate oligonucleotide primers fromthe amino acid sequence known for other nuclear receptors which arerelated to the rhAR protein; (5) screening a rhAR-containing cDNAlibrary constructed in a bacteriophage or plasmid shuttle vector with apartial cDNA encoding the rhAR protein. This strategy may also involveusing gene-specific oligonucleotide primers for PCR amplification ofrhAR cDNA identified as an EST as described above; or (6) designing 5′and 3′ gene specific oligonucleotides using SEQ ID NO:1 or 3 as atemplate so that either the full-length cDNA may be generated by knownPCR techniques, or a portion of the coding region may be generated bythese same known PCR techniques to generate and isolate a portion of thecoding region to use as a probe to screen one of numerous types of cDNAand/or genomic libraries in order to isolate a full-length version ofthe nucleotide molecule encoding rhAR.

It is readily apparent to those ordinarily skilled in the art that othertypes of libraries, as well as libraries constructed from other celltypes-or species types, may be useful for isolating a rhAR-encoding DNAor a rhAR homologue. Other types of libraries include, but are notlimited to, cDNA libraries derived from other cells or cell lines otherthan rhAR cells or tissue such as murine cells, rodent cells or anyother such vertebrate host which may contain rhAR-encoding DNA.Additionally a rhAR gene and homologues may be isolated byoligonucleotide- or polynucleotide-based hybridization screening of avertebrate genomic library, including but not limited to, a murinegenomic library, a rodent genomic library, as well as concomitant rhARgenomic DNA libraries.

It is readily apparent to those skilled in the art that suitable cDNAlibraries may be prepared from cells or cell lines which have rhARactivity. The selection of cells or cell lines for use in preparing acDNA library to isolate a cDNA encoding rhAR may be done by firstmeasuring cell-associated rhAR activity using any known assay availablefor such a purpose.

Preparation of cDNA libraries can be performed by standard techniqueswell known in the art. Well known cDNA library construction techniquescan be found for example, in Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. Complementary DNA libraries may also be obtained from numerouscommercial sources, including but not limited to Clontech Laboratories,Inc. and Stratagene.

It is also readily apparent to those skilled in the art that DNAencoding rhAR may also be isolated from a suitable genomic DNA library.Construction of genomic DNA libraries can be performed by standardtechniques well known in the art. Well known genomic DNA libraryconstruction techniques can be found in Sambrook, et al., supra.

In order to clone the rhAR gene by one of the preferred methods, theamino acid sequence or DNA sequence of rhAR or a homologous protein maybe necessary. To accomplish this, the rhAR protein or a homologousprotein may be purified and partial amino acid sequence determined byautomated sequenators or mass spectroscopy. It is not necessary todetermine the entire amino acid sequence, but the linear sequence of tworegions of 6 to 8 amino acids can be determined for the PCRamplification of a partial rhAR DNA fragment. Once suitable amino acidsequences have been identified, the DNA molecules capable of encodingthem are synthesized. Because the genetic code is degenerate, more thanone codon may be used to encode a particular amino acid, and therefore,the amino acid sequence can be encoded by any of a set of similar DNAoligonucleotides. Only one member of the set will be identical to therhAR sequence but others in the set will be capable of hybridizing torhAR DNA even in the presence of DNA oligonucleotides with mismatches.The mismatched DNA oligonucleotides may still sufficiently hybridize tothe rhAR DNA to permit identification and isolation of rhAR encodingDNA. Alternatively, the nucleotide sequence of a region of an expressedsequence may be identified by searching one or more available genomicdatabases. Gene-specific primers may be used to perform PCRamplification of a cDNA of interest from either a cDNA library or apopulation of cDNAs. As noted above, the appropriate nucleotide sequencefor use in a PCR-based method may be obtained from SEQ ID NO: 1 or18–20, either for the purpose of isolating overlapping 5′ and 3′ RACEproducts for generation of a full-length sequence coding for rhAR, or toisolate a portion of the nucleotide molecule coding for rhAR for use asa probe to screen one or more cDNA- or genomic-based libraries toisolate a full-length molecule encoding rhAR or rhAR-like proteins.

In an exemplified method, the rhAR full-length cDNA of the presentinvention was isolated by screening template cDNA synthesized fromMacaca mulatta prostate mRNA. Oligonucleotide primers based on Macacafascicularis AR were synthesized. Template cDNA was synthesized fromMacaca mulatta prostate mRNA. NH₂ portion and COOH-portion primer pairswere used to generate two PCR fragments, which were subcloned,characterized and assembled into a full length DNA sequence (see SEQ IDNOs: 1 and 3). The cloned Macaca mulatta AR cDNA has 7 nucleotidedifferences from Macaca fascicularis AR in the coding region whichresult in two amino acid residues difference (FIG. 4). The two macaquepolyQ and polyG sequences are identical to each other, and are in turnshorter than the corresponding human sequences. A single amino aciddifference between the macaque and human AR, [Ala-632], is present inthe DBD-Hinge-LBD region.

The present invention also relates to recombinant vectors andrecombinant hosts, both prokaryotic and eukaryotic, which have beentransfected and/or transformed with the nucleic acid molecules disclosedthroughout this specification.

The present invention also relates to methods of expressing rhAR andbiological equivalents disclosed herein, the expressed, processed formof the protein, assays employing these recombinantly expressed geneproducts, cells expressing these gene products, and agonistic and/orantagonistic compounds identified through the use of assays utilizingthese recombinant forms, including, but not limited to, one or moremodulators of rhAR, either through direct contact with the LBD orthrough direct or indirect contact with a ligand which either interactswith the DBD or with the wild-type transcription complex which theandrogen receptor interacts in trans, thereby modulating bone biology,for example.

The present invention relates to methods of expressing rhAR inrecombinant systems and of identifying agonists and antagonists of rhAR.The novel rhAR proteins of the present invention are suitable for use inan assay procedure for the identification of compounds which modulatethe transactivation activity of mammalian rhAR. Modulating rhARactivity, as described herein includes the inhibition or activation ofthis soluble transacting factor and therefore includes directly orindirectly affecting the normal regulation of the rhAR activity.Compounds that modulate rhAR include agonists, antagonists and compoundswhich directly or indirectly affect regulation of rhAR. When screeningcompounds in order to identify potential pharmaceuticals thatspecifically interact with a target protein, it is necessary to ensurethat the compounds identified are as specific as possible for the targetprotein. To do this, it may necessary to screen the compounds against aswide an array as possible of proteins that are similar to the targetreceptor, including species homologous to rhesus androgen receptor.Thus, in order to find compounds that are potential pharmaceuticals thatinteract with rhAR, it is necessary not only to ensure that thecompounds interact with rhAR (the “plus target”) and produce the desiredpharmacological effect through rhAR, it is also necessary to determinethat the compounds do not interact with proteins B, C, D, etc. (the“minus targets”). In general, as part of a screening program, it isimportant to have as many minus targets as possible (see Hodgson, 1992,Bio/Technology 10:973–980, @ 980). rhAR proteins and the DNA moleculesencoding this protein may serve this purpose in assays utilizing, forexample, other members of the nuclear receptor superfamily.

As used herein, a “biologically functional derivative” of a wild-typerhAR possesses a biological activity that is related to the biologicalactivity of the wild type rhAR. The term “functional derivative” isintended to include the “fragments,” “mutants,” “variants,” “degeneratevariants,” “analogs” and “homologues” of the wild type rhAR protein. Theterm “fragment” is meant to refer to any polypeptide subset of wild-typerhAR, including but not necessarily limited to rhAR proteins comprisingamino acid substitutions, deletions, additions, amino terminaltruncations and/or carboxy-terminal truncations. The term “mutant” ismeant to refer a subset of a biologically active fragment that may besubstantially similar to the wild-type form but possesses distinguishingbiological characteristics. Such altered characteristics include but arein no way limited to altered substrate binding, altered substrateaffinity and altered sensitivity to chemical compounds affectingbiological activity of the rhAR or a rhAR functional derivative. Theterm “variant” is meant to refer to a molecule substantially similar instructure and function to either the wild-type protein or to a fragmentthereof.

A variety of mammalian expression vectors may be used to expressrecombinant rhAR in mammalian cells. Expression vectors are definedherein as DNA sequences that are required for the transcription ofcloned DNA and the translation of their mRNAs in an appropriate host.Such vectors can be used to express eukaryotic DNA in a variety of hostssuch as bacteria, blue green algae, plant cells, insect cells and animalcells. Specifically designed vectors allow the shuttling of DNA betweenhosts such as bacteria-yeast or bacteria-animal cells. An appropriatelyconstructed expression vector should contain: an origin of replicationfor autonomous replication in host cells, selectable markers, a limitednumber of useful restriction enzyme sites, a potential for high copynumber, and active promoters. A promoter is defined as a DNA sequencethat directs RNA polymerase to bind to DNA and initiate RNA synthesis. Astrong promoter is one that causes mRNAs to be initiated at highfrequency. Expression vectors may include, but are not limited to,cloning vectors, modified cloning vectors, specifically designedplasmids or viruses.

Commercially available mammalian expression vectors which may besuitable for recombinant rhAR expression, include but are not limitedto, pcDNA3.1 (Invitrogen), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39(New England Bioloabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3(Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5(Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2) (ATCC 37110),pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and IZD35 (ATCC37565).

A variety of bacterial expression vectors may be used to expressrecombinant rhAR in bacterial cells. Commercially available bacterialexpression vectors which may be suitable for recombinant rhAR expressioninclude, but are not limited to pCRII (Invitrogen), pCR2.1 (Invitrogen),pQE (Qiagen), pET11a (Novagen), lambda gt11 (Invitrogen), pKK223-3(Pharmacia), and pGEX2T (Pharmacia).

A variety of fungal cell expression vectors may be used to expressrecombinant rhAR in fungal cells. Commercially available fungal cellexpression vectors which may be suitable for recombinant rhAR expressioninclude but are not limited to the ESP® yeast expression system, whichutilizes S. pombe as the expression host, pYES2 (Invitrogen) and Pichiaexpression vector (Invitrogen).

A variety of insect cell expression vectors may be used to expressrecombinant receptor in insect cells. Commercially available insect cellexpression vectors which may be suitable for recombinant expression ofrhAR include but are not limited to pBlueBacIII and pBlueBacHis2(Invitrogen), and pAcG2T (Pharmingen).

An expression vector containing DNA encoding a rhAR or rhAR-like proteinmay be used for expression of rhAR in a recombinant host cell.Recombinant host cells may be prokaryotic or eukaryotic, including butnot limited to bacteria such as E. coli, fungal cells such as yeast,mammalian cells including but not limited to cell lines of rhAR, bovine,porcine, monkey and rodent origin, and insect cells including but notlimited to Drosophila- and silkworm-derived cell lines. Cell linesderived from mammalian species which may be suitable and which arecommercially available, include but are not limited to, L cells L-M(TK⁻) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), Saos-2 (ATCC HTB-85),293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCCCRL 1650), COS-7 (ATCC CRL 1651), CHO-K1(ATCC CCL 61), 3T3 (ATCC CCL92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616),BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171) and CPAE (ATCC CCL 209).

The expression vector may be introduced into host cells via any one of anumber of techniques including but not limited to transfection,transformation, protoplast fusion, and electroporation. The expressionvector-containing cells are individually analyzed to determine whetherthey produce rhAR protein. Identification of rhAR expressing cells maybe done by several means, including but not limited to immunologicalreactivity with anti-rhAR antibodies, labeled ligand binding and thepresence of host cell-associated rhAR activity.

The cloned rhAR cDNA obtained through the methods described above may berecombinantly expressed by molecular cloning into an expression vector(such as pcDNA3.1, pQE, pBlueBacHis2 and pLITMUS28) containing asuitable promoter and other appropriate transcription regulatoryelements, and transferred into prokaryotic or eukaryotic host cells toproduce recombinant rhAR. Techniques for such manipulations can be founddescribed in Sambrook, et al., supra , are discussed at length in theExample section and are well known and easily available to the artisanof ordinary skill in the art.

Expression of rhAR DNA may also be performed using in vitro producedsynthetic mRNA. Synthetic mRNA can be efficiently translated in variouscell-free systems, including but not limited to wheat germ extracts andreticulocyte extracts, as well as efficiently translated in cell basedsystems, including but not limited to microinjection into frog oocytes,with microinjection into frog oocytes being preferred.

To determine the rhAR cDNA sequence(s) that yields optimal levels ofrhAR, cDNA molecules including but not limited to the following can beconstructed: a cDNA fragment containing the full-length open readingframe for rhAR as well as various constructs containing portions of thecDNA encoding only specific domains of the protein or rearranged domainsof the protein. All constructs can be designed to contain none, all orportions of the 5′ and/or 3′ untranslated region of a rhAR cDNA. Theexpression levels and activity of rhAR can be determined following theintroduction, both singly and in combination, of these constructs intoappropriate host cells. Following determination of the rhAR cDNAcassette yielding optimal expression in transient assays, this rhAR cDNAconstruct is transferred to a variety of expression vectors (includingrecombinant viruses), including but not limited to those for mammaliancells, plant cells, insect cells, oocytes, bacteria, and yeast cells.

A preferred aspect of the present invention relates to a substantiallypurified form of the novel nuclear trans-acting receptor protein, arhesus androgen receptor protein, which is disclosed in FIG. 2 (SEQ IDNO:2) as well as a polymorph of the protein disclosed in SEQ ID NO:2,disclosed herein as SEQ ID NO:4.

The rhAR protein disclosed in SEQ ID NO:2 is as follows:

MEVQLGLGRV YPRPPSKTYR GAFQNLFQSV REVIQNPGPR HPEAASAAPP (SEQ ID NO: 2)GASLQQQQQQ QQETSPRQQQ QQQQGEDGSP QAHRRGPTGY LVLDEEQQPS QPQSAPECHPERGCVPEPGA AVAAGKGLPQ QLPAPPDEDD SAAPSTLSLL GPTFPGLSSC SADLKDILSEASTMQLLQQQ QQEAVSEGSS SGRAREASGA PTSSKDNYL E GTSTISDSAK ELCKAVSVSMGLGVEALEHL SPGEQLRGDC MYAPVLGVPP AVRPTPCAPL AECKGSLLDD SAGKSTEDTAEYSPFKGGYT KGLEGESLGC SGSAAAGSSG TLELPSTLSL YKSGALDEAA AYQSRDYYNFPLALAGPPPP PPPPHPHARI KLENPLDYGS AWAAAAAQCR YGDLASLHGA GAAGPGSGSPSAAASSSWHT LFTAEEGQLY GPCGGGGGGG GGGGGGAGEA GAVAPYGYTR PPQGLAGQEGDFTAPDVWYP GGMVSRVPYP SPTCVKSEMG PWMDSYSGPY GDMRLETARD HVLPIDYYFP PQKTCLICGD EASGCHYGAL TCGSCKVFFK RAAEGKQKYL CASRNDCTID KFRRKNCPSC RLRKCYEAGMTLGARKLKKL GNLKLQEEGE ASSTTSPTEE TAQKLTVSHI EGYECQPIFL NVLEAIEPGVVCAGHDNNQP DSFAALLSSL NELGERQLVH VVKWAKALPG FRNLHVDDQM AVIQYSWMGLMVFAMGWRSF TNVNSRMLYF APDLVFNEYR MHKSRMYSQC VRMRHLSQEF GWLQITPQEFLCMKALLLFS IIPVDGLKNQ KFFDELRMNY IKELDRIIAC KRKNPTSCSR RFYQLTKLLDSVQPIARELH QFTFDLLIKS HMVSVDFPEM MAEIISVQVP KILSGKVKPI YFHTQ.

As noted herein, the Glu-210 residue (underlined and bolded) of rhAR ofSEQ ID NO:2 represents an allelic variant at nucleotide 1051 of SEQ IDNO:1. A single nucleotide change at nucleotide 1051 from ‘A’ to ‘G’results in an amino acid change at residue 210 of the rhAR, from the Gluresidue of SEQ ID NO:2 to a Gly residue (underlined and bolded), shownbelow as SEQ ID NO:4:

MEVQLGLGRV YPRPPSKTYR GAFQNLFQSV REVIQNPGPR HPEAASAAPP (SEQ ID NO: 4)GASLQQQQQQ QQETSPRQQQ QQQQGEDGSP QAHRRGPTGY LVLDEEQQPS QPQSAPECHPERGCVPEPGA AVAAGKGLPQ QLPAPPDEDD SAAPSTLSLL GPTFPGLSSC SADLKDILSEASTMQLLQQQ QQEAVSEGSS SGRAREASGA PTSSKDNYL G GTSTISDSAK ELCKAVSVSMGLGVEALEHL SPGEQLRGDC MYAPVLGVPP AVRPTPCAPL AECKGSLLDD SAGKSTEDTAEYSPFKGGYT KGLEGESLGC SGSAAAGSSG TLELPSTLSL YKSGALDEAA AYQSRDYYNFPLALAGPPPP PPPPHPHARI KLENPLDYGS AWAAAAAQCR YGDLASLHGA GAAGPGSGSPSAAASSSWHT LFTAEEGQLY GPCGGGGGGG GGGGGGAGEA GAVAPYGYTR PPQGLAGQEGDFTAPDVWYP GGMVSRVPYP SPTCVKSEMG PWMDSYSGPY GDMRLETARD HVLPIDYYFP PQKTCLICGD EASGCHYGAL TCGSCKVFFK RAAEGKQKYL CASRNDCTID KFRRKNCPSC RLRKCYEAGMTLGARKLKKL GNLKLQEEGE ASSTTSPTEE TAQKLTVSHI EGYECQPIFL NVLEAIEPGVVCAGHDNNQP DSFAALLSSL NELGERQLVH VVKWAKALPG FRNLHVDDQM AVIQYSWMGLMVFAMGWRSF TNVNSRMLYF APDLVFNEYR MHKSRMYSQC VRMRHLSQEF GWLQITPQEFLCMKALLLFS IIPVDGLKNQ KFFDELRMNY IKELDRIIAC KRKNPTSCSR RFYQLTKLLDSVQPIARELH QFTFDLLIKS HMVSVDFPEM MAEIISVQVP KILSGKVKPI YFHTQ.The underlined portions of SEQ ID NOs:2 and 4, from amino acid residue535 to residue 600, represent the DNA binding domain (DBD) of the rhARreceptor protein. The DBD participates in regulating protein-proteininteractions in AR transrepression pathway. Aarnisalo et al.,Endocrinology 140(7):3097 (1999). Transcription activation andrepression functions of the androgen receptor are differentiallyinfluenced by mutations in the DNA-binding domain. In transactivation,AR forms homodimer and binds DNA response element via DBD.

The present invention also relates to a substantially purified, fullyprocessed (including proteolytic processing, such as processing of anatural, hybrid or synthetic signal sequence, glycosylation and/orphosphorylation) mature rhAR protein obtained from a recombinant hostcell containing a DNA expression vector comprising a nucleotide sequenceas set forth in SEQ ID NOs: 1 and 3, or nucleic acid fragments thereofas described above, such DNA expression vectors expressing therespective rhAR protein or rhAR precursor protein. It is especiallypreferred that the recombinant host cell be a eukaryotic host cell,including but not limited to a mammalian cell line or an insect cellline. In another embodiment, it is especially preferred that therecombinant host cell be a yeast host cell.

The present invention also relates to isolated nucleic acid moleculeswhich are fusion constructions expressing fusion proteins useful inassays to identify compounds which modulate mammalian AR. A preferredaspect of this portion of the invention includes, but is not limited to,glutathione S-transferase GST-rhAR fusion constructs. These fusionconstructs include, but are not limited to, all or a portion of theligand-binding domain of rhAR, respectively, as an in-frame fusion atthe carboxy terminus of the GST gene. The disclosure of SEQ ID NOS: 1and 3 provide the artisan of ordinary skill the information necessary toconstruct any such nucleic acid molecule encoding a GST-nuclear receptorfusion protein. Soluble recombinant GST-nuclear receptor fusion proteinsmay be expressed in various expression systems, including but in nowmanner limited to a yeast expression system (see Example Section 2), orSpodoptera frugiperda (Sf21) within insect cells (Invitrogen) using abaculovirus expression vector (e.g., Bac-N-Blue DNA from Invitrogen orpAcG2T from Pharmingen). Example Section 2 discloses construction ofGST-Flag-rhARLBD (Mr=60 kDa), which is expressed in yeast. This fusionprotein is purified by standard techniques and used in a hydoxyapatitebinding assay in the presence of labeled R1881 and unlabeled testcompounds. After a parallel binding reaction where increasingconcentration of unlabeled test compounds are incubated with ³H-R1881, ahydroxyapatite slurry is prepared and processed. Unbound ligand isremoved and the subsequent hydroxyapatite pellet is washed and ligandbound GST-rhAR is assessed to quantify the amount of radioligand(³H-R1881) bound to the recombinant rhAR fusion protein. Results arecompared to known high affinity ligands such as 5-alphadihydrotestosterone and unlabeled R1881, which exhibit IC50s of ca. 1nM. See, Asselin and Melancon, 1977, Steroids 30: 591–604; Ghanadian etal., 1977, Urol. Res. 5(4): 169–173.

Other assays are contemplated for the rhAR cDNA clones of the presentinvention, including but not limited to the use of these clone(s) to setup co-transfection assays to measure bioactivity of compounds, or toset-up mammalian two-hybrid assays to test the effect of compounds on N—and C-terminus interaction of Macaca mulatta AR.

For example, the present invention relates to constructs wherein areceptor construct (e.g., containing the rhAR LBD, e.g., Gal4-rhAR-LBD)and a reporter construct (such as SEAP or LacZ) with regulatory sitesthat respond to increases and decreases in expression of the receptorconstruct. Therefore, the present invention includes assays by whichmodulators of rhAR are identified. Methods for identifying agonists andantagonists of other receptors are well known in the art and can beadapted to identify compounds which effect in vivo levels of rhAR.Accordingly, the present invention includes a method for determiningwhether a substance is a potential modulator of AR levels thatcomprises:

(a) transfecting or transforming cells with an expression vectorencoding rhAR, (such as the LBD of rhAR) also known as the receptorvector;

(b) transfecting or transforming the cells of step (a) with secondexpression vector, also known as a reporter vector, which comprises anelement known to respond to rhAR through protein-protein interactionsbut bind a non-rhAR protein or a promoter fragment fused upstream of areporter gene;

(c) allowing the transfected cells to grow for a time sufficient forrhAR to be expressed;

(d) exposing some of the transfected cells expressing rhAR, the “testcells” to a test substance while not exposing control cells to the testsubstance;

(e) measuring the expression of the reporter gene in both the test cellsand control cells.

Of course, “controls” in such assays may take many forms, such as butnot limited to the recitation of step (d) above, or possibly the use ofcells not transfected with the nucleic acid molecule expressing rhAR(i.e., non-transfected cells), or cells transfected with vector alone,minus the coding region for rhAR. Also, conditions under which step (d)of the method is practiced are conditions that are typically used in theart for the study of protein-ligand interactions: e.g., physiologicalpH; salt conditions such as those represented by such commonly usedbuffers as PBS or in tissue culture media; a temperature of about 4° C.to about 55° C. This assay may be conducted with crude cell lysate, orwith more purified materials. Alternatively, the transrepression assaymay be carried out as follows:

(a) provide test cells by transfecting cells with a receptor expressionvector that directs the expression of rhAR or a portion thereof (such asthe LBD of rhAR) in the cells;

(b) providing test cells by transfecting the cells of step (a) with asecond reporter expression vector that directs expression of a reportergene under control of a regulatory element which is responsive to rhARvia protein-protein interactions or a portion of the rhAR construct;

(c) exposing the test cells to the substance;

(d) measuring expression of the reporter gene;

(e) comparing the amount of expression of the reporter gene in the testcells with the amount of expression of the reporter gene in controlcells that have been transfected with a reporter vector of step (b) butnot a receptor vector of step (a).

This assay may be conducted with transfected mammalian cell lines usingcell-permeable test compounds.

An alternative assay would be one wherein multiple receptor/reporterconstructs are transfected into cells such that the general nature ofthe trans-acting factor can be measured. It is evident that any numberof variations known to one of skill in the art may be utilized in orderto provide for an assay to measure the effect of a substance on theability of the nuclear receptor proteins of the present invention toeffect transcription of a promoter of interest via protein-proteininteractions with heterologous DNA binding proteins.

The present invention includes additional methods for determiningwhether a substance is capable of binding to rhAR, i.e., whether thesubstance is a potential agonist or an antagonist of rhAR, where themethod comprises:

(a) providing test cells by transfecting cells with an expression vectorthat directs the expression of rhAR in the cells;

(b) exposing the test cells and control cells to the substance;

(c) measuring the amount of binding of the substance to rhAR;

(d) comparing the amount of binding of the substance to rhAR in the testcells with the amount of binding of the substance to control cells thathave not been transfected with rhAR or a portion thereof; wherein if theamount of binding of the substance is greater in the test cells ascompared to the control cells, the substance is capable of binding torhAR. Determining whether the substance is actually an agonist orantagonist can then be accomplished by the use of functional assays suchas the transrepression assay as described above.

Test compounds that regulate rhAR function through gene expression maybe evaluated employing the method above.

The conditions under which step (b) of the method is practiced areconditions that are typically used in the art for the study ofprotein-ligand interactions: e.g., physiological pH; salt conditionssuch as those represented by such commonly used buffers as PBS or intissue culture media; a temperature of about 4° C. to about 55° C.

The assays described above can be carried out with cells that have beentransiently or stably transfected with rhAR. Transfection is meant toinclude any method known in the art for introducing rhAR into the testcells. For example, transfection includes calcium phosphate or calciumchloride mediated transfection, lipofection, infection with a retroviralconstruct containing rhAR, and electroporation. Where binding of thesubstance or agonist to rhAR is measured, such binding can be measuredby employing a labeled substance or agonist. The substance or agonistcan be labeled in any convenient manner known to the art, e.g.,radioactively, fluorescently, enzymatically.

The rhAR of the present invention may be used to screen for rhAR ligandsby assessing transcriptional regulation proceeding via the ligand-boundrhAR-transcription factor protein-protein interactions. Alternatively,the rhAR of the present invention may be employed to screen for rhARligands using co-transfection with classical nuclear receptor responseelements that bind the rhAR DBD.

The present invention also relates to polyclonal and monoclonalantibodies raised in response to rhAR. Recombinant rhAR protein can beseparated from other cellular proteins by use of an immunoaffinitycolumn made with monoclonal or polyclonal antibodies specific forfull-length rhAR protein, or polypeptide fragments of rhAR protein.Additionally, polyclonal or monoclonal antibodies may be raised againsta synthetic peptide (usually from about 9 to about 25 amino acids inlength) from a portion of the protein as disclosed in SEQ ID NO:2 and/orSEQ ID NO:4. Monospecific antibodies to rhAR are purified from mammalianantisera containing antibodies reactive against rhAR or are prepared asmonoclonal antibodies reactive with rhAR using the technique of Kohlerand Milstein (1975, Nature 256: 495–497). Monospecific antibody as usedherein is defined as a single antibody species or multiple antibodyspecies with homogenous binding characteristics for rhAR. Homogenousbinding as used herein refers to the ability of the antibody species tobind to a specific antigen or epitope, such as those associated withrhAR, as described above. rhAR-specific antibodies are raised byimmunizing animals such as mice, rats, guinea pigs, rabbits, goats,horses and the like, with an appropriate concentration of rhAR proteinor a synthetic peptide generated from a portion of rhAR with or withoutan immune adjuvant.

Preimmune serum is collected prior to the first immunization. Eachanimal receives between about 0.1 mg and about 1000 mg of rhAR proteinassociated with an acceptable immune adjuvant. Such acceptable adjuvantsinclude, but are not limited to, Freund's complete, Freund's incomplete,alum-precipitate, water in oil emulsion containing Corynebacteriumparvum and tRNA. The initial immunization consists of rhAR protein orpeptide fragment thereof in, preferably, Freund's complete adjuvant atmultiple sites, either subcutaneously (SC), intraperitoneally (IP) orboth. Each animal is bled at regular intervals, preferably weekly, todetermine antibody titer. The animals may or may not receive boosterinjections following the initial immunization. Those animals receivingbooster injections are generally given an equal amount of rhAR inFreund's incomplete adjuvant by the same route. Booster injections aregiven at about three week intervals until maximal titers are obtained.At about 7 days after each booster immunization or about weekly after asingle immunization, the animals are bled, the serum collected, andaliquots are stored at about −20° C.

Monoclonal antibodies (mAb) reactive with rhAR are prepared byimmunizing inbred mice, preferably Balb/c, with rhAR protein. The miceare immunized by the IP or SC route with about 1 mg to about 100 mg,preferably about 10 mg, of rhAR protein in about 0.5 ml buffer or salineincorporated in an equal volume of an acceptable adjuvant, as discussedabove. Freund's complete adjuvant is preferred. The mice receive aninitial immunization on day 0 and are rested for about 3 to about 30weeks. Immunized mice are given one or more booster immunizations ofabout 1 to about 100 mg of rhAR in a buffer solution such as phosphatebuffered saline by the intravenous (IV) route. Lymphocytes, fromantibody positive mice, preferably splenic lymphocytes, are obtained byremoving spleens from immunized mice by standard procedures known in theart. Hybridoma cells are produced by mixing the splenic lymphocytes withan appropriate fusion partner, preferably myeloma cells, underconditions that will allow the formation of stable hybridomas. Fusionpartners may include, but are not limited to: mouse myelomas P3/NS1/Ag4-1, MPC-11, S-194 and Sp 2/0, with Sp 2/0 being preferred. The antibodyproducing cells and myeloma cells are fused in polyethylene glycol,about 1000 mol. wt., at concentrations from about 30% to about 50%.Fused hybridoma cells are selected by growth in hypoxanthine, thymidineand aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) byprocedures known in the art. Supernatant fluids are collected formgrowth positive wells on about days 14, 18, and 21 and are screened forantibody production by an immunoassay such as solid phaseimmunoradioassay (SPIRA) using rhAR as the antigen. The culture fluidsare also tested in the Ouchterlony precipitation assay to determine theisotype of the mAb. Hybridoma cells from antibody positive wells arecloned by a technique such as the soft agar technique of MacPherson,1973, Soft Agar Techniques, in Tissue Culture Methods and Applications,Kruse and Paterson, Eds., Academic Press.

Monoclonal antibodies are produced in vivo by injection of pristineprimed Balb/c mice, approximately 0.5 ml per mouse, with about 2×10⁶ toabout 6×10⁶ hybridoma cells about 4 days after priming. Ascites fluid iscollected at approximately 8–12 days after cell transfer and themonoclonal antibodies are purified by techniques known in the art.

In vitro production of anti-rhAR mAb is carried out by growing thehybridoma in DMEM containing about 2% fetal calf serum to obtainsufficient quantities of the specific mAb. The mAb are purified bytechniques known in the art.

Antibody titers of ascites or hybridoma culture fluids are determined byvarious serological or immunological assays which include, but are notlimited to, precipitation, passive agglutination, enzyme-linkedimmunosorbent antibody (ELISA) technique and radioimmunoassay (RIA)techniques. Similar assays are used to detect the presence of human rhARin body fluids or tissue and cell extracts.

It is readily apparent to those skilled in the art that theabove-described methods for producing monospecific antibodies may beutilized to produce antibodies specific for rhAR peptide fragments, orfull-length rhAR.

rhAR antibody affinity columns are made, for example, by adding theantibodies to Affigel-10 (Biorad), a gel support which is pre-activatedwith N-hydroxysuccinimide esters such that the antibodies form covalentlinkages with the agarose gel bead support. The antibodies are thencoupled to the gel via amide bonds with the spacer arm. The remainingactivated esters are then quenched with 1M ethanolamine HCl (pH 8.0).The column is washed with water followed by 0.23 M glycine HCl (pH 2.6)to remove any non-conjugated antibody or extraneous protein. The columnis then equilibrated in phosphate buffered saline (PBS) (pH 7.3) and thecell culture supernatants or cell extracts containing full-length rhARor rhAR protein fragments are slowly passed through the column. Thecolumn is then washed with phosphate buffered saline until the opticaldensity (A280) falls to background, then the protein is eluted with 0.23M glycine-HCl (pH 2.6). The purified rhAR protein is then dialyzedagainst phosphate buffered saline.

Levels of rhAR in host cells are quantified by a variety of techniquesincluding, but not limited to, immunoaffinity and/or ligand affinitytechniques. rhAR-specific affinity beads or rhAR-specific antibodies areused to isolate ³⁵S-methionine labeled or unlabelled rhAR. Labeled rhARprotein is analyzed by SDS-PAGE. Unlabelled rhAR protein is detected byWestern blotting, ELISA or RIA assays employing either rhAR proteinspecific antibodies and/or antiphosphotyrosine antibodies.

Following expression of rhAR in a host cell, rhAR protein may berecovered to provide rhAR protein in active form. Several rhAR proteinpurification procedures are available and suitable for use. RecombinantrhAR protein may be purified from cell lysates and extracts, or fromconditioned culture medium, by various combinations of, or individualapplication of salt fractionation, ion exchange chromatography, sizeexclusion chromatography, hydroxylapatite adsorption chromatography andhydrophobic interaction chromatography.

The DNA molecules, RNA molecules, recombinant protein and antibodies ofthe present invention may be used to screen and measure levels of rhAR.The recombinant proteins, DNA molecules, RNA molecules and antibodieslend themselves to the formulation of kits suitable for the detectionand typing of rhAR. Such a kit would comprise a compartmentalizedcarrier suitable to hold in close confinement at least one container.The carrier would further comprise reagents such as recombinant rhAR oranti-rhAR antibodies suitable for detecting rhAR. The carrier may alsocontain a means for detection such as labeled antigen or enzymesubstrates or the like.

Pharmaceutically useful compositions comprising modulators of rhAR maybe formulated according to known methods such as by the admixture of apharmaceutically acceptable carrier. Examples of such carriers andmethods of formulation may be found in Remington's PharmaceuticalSciences. To form a pharmaceutically acceptable composition suitable foreffective administration, such compositions will contain an effectiveamount of the protein, DNA, RNA, modified rhAR, or either rhAR agonistsor antagonists.

Therapeutic or diagnostic compositions comprising modulators of rhAR areadministered to an individual in amounts sufficient to treat or diagnosedisorders. The effective amount may vary according to a variety offactors such as the individual's condition, weight, sex and age. Otherfactors include the mode of administration.

The pharmaceutical compositions may be provided to the individual by avariety of routes such as subcutaneous, topical, oral and intramuscular.

The term “chemical derivative” describes a molecule that containsadditional chemical moieties that are not normally a part of the basemolecule. Such moieties may improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties mayattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule. Examples of such moieties are describedin a variety of texts, such as Remington's Pharmaceutical Sciences.

Compounds identified according to the methods disclosed herein may beused alone at appropriate dosages. Alternatively, co-administration orsequential administration of other agents may be desirable.

The present invention also has the objective of providing suitabletopical, oral, systemic and parenteral pharmaceutical formulations foruse in the novel methods of treatment of the present invention. Thecompositions containing compounds identified according to this inventionas the active ingredient can be administered in a wide variety oftherapeutic dosage forms in conventional vehicles for administration.For example, the compounds can be administered in such oral dosage formsas tablets, capsules (each including timed release and sustained releaseformulations), pills, powders, granules, elixirs, tinctures, solutions,suspensions, syrups and emulsions, or by injection. Likewise, they mayalso be administered in intravenous (both bolus and infusion),intraperitoneal, subcutaneous, topical with or without occlusion, orintramuscular form, all using forms well known to those of ordinaryskill in the pharmaceutical arts.

Advantageously, compounds of the present invention may be administeredin a single daily dose, or the total daily dosage may be administered individed doses of two, three or four times daily. Furthermore, compoundsfor the present invention can be administered in intranasal form viatopical use of suitable intranasal vehicles, or via transdermal routes,using those forms of transdermal skin patches well known to those ofordinary skill in that art. To be administered in the form of atransdermal delivery system, the dosage administration will, of course,be continuous rather than intermittent throughout the dosage regimen.

For combination treatment with more than one active agent, where theactive agents are in separate dosage formulations, the active agents canbe administered concurrently, or they each can be administered atseparately staggered times.

The dosage regimen utilizing the compounds of the present invention isselected in accordance with a variety of factors including type,species, age, weight, sex and medical condition of the patient; theseverity of the condition to be treated; the route of administration;the renal, hepatic and cardiovascular function of the patient; and theparticular compound thereof employed. A physician or veterinarian ofordinary skill can readily determine and prescribe the effective amountof the drug required to prevent, counter or arrest the progress of thecondition. Optimal precision in achieving concentrations of drug withinthe range that yields efficacy without toxicity requires a regimen basedon the kinetics of the drugs availability to target sites. This involvesa consideration of the distribution, equilibrium, and elimination of adrug.

The following examples are provided to illustrate the present inventionwithout, however, limiting the same hereto.

EXAMPLE 1 Isolation and Characterization of a DNA Molecule Encoding rhAR

The DNA sequence for Macaca fascicularis monkey AR (Gen Bank Acc. #U94179, also disclosed in the attached sequence listing as SEQ ID NO:6)and an EST for Macaca mulatta AR (Gen Bank Accession No. AF092930) maybe used for primer designing. The nucleotide sequence for Macaca mulattaAR EST is as follows:

TCTCAAGAGT TTGGATGGCT CCAAATCACC CCCCAGGAAT TCCTGTGCAT (SEQ ID NO: 7)GAAAGCGCTG CTACTCTTCA GCATTATTCC AGTGGATGGG CTGAAAAATC AAAAATTCTTTGATGAACTT CGAATGAACT ACATCAAGGA ACTCGATCGT ATCATTGCAT GCAAAAGAAAAAATCCCACA TCCTGCTCAA GGCGTTTCTA CCAGCTCACC AAGCTCCTGG ACTCCGTGCAGCCTATTGCG AGAGAGCTGC ATCAGTTCAC TTTTGACCTG CTAATCAAGT CACACATGGTGAGCGTGGAC TTTCCGGAAA TGATGGCAGA GATCATCTC.

Messenger RNA from rhesus monkey prostate was prepared and cDNA wassynthesized by standard methods. The full-length Macaca mulatta AR wascloned via standard PCR methodology. Oligonucleotide primers were basedon Macaca fascicularis AR. Template cDNA was synthesized from Macacamulatta prostate mRNA. Primer pairs mkARF2 (5′-ATG GAG GTG CAG TTA GGGCTG-3′; SEQ ID NO:8) and mkARR5 (5′-GGT CTT CTG GGG TGG AAA GTA-3′; SEQID NO:9) were used to obtain the NH₂-terminal portion of the gene viaPCR, while the COOH-terminal portion was obtained using mkARF5 (5′-ACGGCT ACA CTC GGC CAC CTC-3′; SEQ ID NO:10) and mkARR2 (5′-AAC AGG CAG AAGACA TCT GAA-3′ SEQ ID NO:11). Each fragment was sub-cloned into a pCRIIvector and sequencing verification was performed on DNA from eachsub-clones. Clones containing wild type cDNA sequences as compared tothe consensus sequence from both NH₂— and COOH— terminal DNA sequenceassembly were used for full-length cDNA construction. The finalfull-length cDNA was obtained through ligating the 5′ and the 3′ end ofthe cDNA at a KpnI site and cloning into a pCRII vector. The nucleotidesequence was again verified via sequencing. Also, the starting Met and5′-UTR information for Macaca mulatta AR was obtained through cDNAextension on subdivided Macaca mulatta cDNA library using mkARR7 primer(5′-GGC GGC CGA GGG TAG ACC CTC-3′ SEQ ID NO:12). The cloned Macacamulatta AR cDNA shows seven nucleotide differences from Macacafascicularis AR in the coding region which result in two amino acidresidues differences. Both open reading frames show identical polyQ andpolyG sequences which are shorter than the human version, with the DBDand LBD regions being identical to the human version.

EXAMPLE 2 Generation of GST-rhAR Fusion Proteins for Use in In VitroScreening Assays

Expression vector construction: PCR fragment containing residues 601 to895, which contains the whole LBD, was inserted into pESP-1 expressionvector (#251600, Stratagene, Lo Jolla, Calif.) at SmaI site which makesthe rhARLBD down stream of GST-Flag tag. The final conjunction sequencesare vector 5′-GGA TCC CCC ACT CTG GGA GCC . . . CTG CCT GTT GGG TAA-3′vector.

AR Expression—GST-Flag-rhARLBD (Mr=60 kDa) is expressed in yeast usingpESP-1 vector according to Stratagene's protocol and lysed inTEGM/DTT/PI buffer [10 mM Tris, pH7.4, 1 mM EDTA, 10% glycerol, 10 mMmolybdate, 2 mM DTT, 50 ul of yeast protease inhibitor cocktail (PI:Sigma) per gram of yeast and 1/10 vol. of PI complete (PI:Boehringer-Mannheim) per gram of yeast.

Fusion Protein Purification—The above fusion protein is purified usinganti-flag M2 affinity gel (Sigma) via batch purification method usingTEGM/DTT buffer. The protein is eluted using TEGM/DTT buffer containing100 ug/ml of Flag peptide.

Hydroxyapatite Binding Assay—Typically, 0.25 ug/ml of recombinantpurified GST-Flag-rhARLBD and 2 nM ³H-R1881 are combined in 100 ulbinding reaction (with 50 mM Tris, pH7.5, 10% glycerol, 0.8 M NaCl, 1mg/ml BSA and 2 mM dithiothreitol) that is incubated for 18 hours at 4°C. ³H-R1881 binding displacement is assessed in parallel bindingreaction aliquots in the presence of varying concentrations of unlabeledtest compounds. Following the initial 18 hour binding reaction, 100 ulof a 50% (wt/vol) hydroxyapatite (HAP) slurry is added to each sample,vortexed, and incubated on ice for ˜10 min. The samples are thencentrifuged and the supernatant aspirated to remove unbound ligand. TheHAP pellet is washed three times with wash buffer (40 mM Tris, pH7.5,100 mM KCl, 1 mM EDTA and 1 mM EGTA). The 3× washed HAP pelletcontaining ligand-bound GST-RhAR is transferred in 95% EtOH to ascintillation vial containing 5 ml scintillation fluid, mixed andcounted to quantify the amount of radioligand (3H-R1881) bound to therecombinant RhAR fusion protein. Results are compared to known highaffinity ligands such as 5-alpha dihydrotestosterone and unlabeledR1881, which exhibit IC50s of ca. 1 nM.

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be understood that the practice of the invention encompasses all ofthe usual variations, adoptions, or modifications, as come within thescope of the following claims and their equivalents.

1. A purified DNA molecule encoding a Macaca mulatta AR protein whereinsaid protein comprises the amino acid sequence as follows: MEVQLGLGRVYPRPPSKTYR GAFQNLFQSV REVIQNPGPR HPEAASAAPP GASLQQQQQQ QQETSPRQQQQQQQGEDGSP QAHRRGPTGY LVLDEEQQPS QPQSAPECHP ERGCVPEPGA AVAAGKGLPQQLPAPPDEDD SAAPSTLSLL GPTFPGLSSC SADLKDILSE ASTMQLLQQQ QQEAVSEGSSSGRAREASGA PTSSKDNYLE GTSTISDSAK ELCKAVSVSM GLGVEALEHL SPGEQLRGDCMYAPVLGVPP AVRPTPCAPL AECKGSLLDD SAGKSTEDTA EYSPFKGGYT KGLEGESLGCSGSAAAGSSG TLELPSTLSL YKSGALDEAA AYQSRDYYNF PLALAGPPPP PPPPHPHARIKLENPLDYGS AWAAAAAQCR YGDLASLHGA GAAGPGSGSP SAAASSSWHT LFTABEGQLYGPCGGGGGGG GGGGGGAGEA GAVAPYGYTR PPQGLAGQEG DFTAPDVWYP GGMVSRVPYPSPTCVKSEMG PWMDSYSGPY GDMRLETAHD HVLPIDYYFP PQKTCLICGD EASGCHYGALTCGSCKVFFK RAAEGKQKYL CASRNDCTID KFRRKIWPSC RLRKCYEAGM TLGARKLKKLGNLKLQEEGE ASSTTSPTEE TAQKLTVSHI EGYECQPIFL NVLEAIEPGV VCAGHDNNQPDSFAALLSSL NELGERQLVH VVKWAKALPG FRNLHVDDQM AVIQYSWNGL MVFAMGWRSFTNVNSRMLYF APDLVFNEYR MHKSRMYSQC VPMRHLSQEF GWLQITPQEF LCMKALLLFSIIPVDGLKNQ KFFDELRMNY IKELDRIIAC KRKNPTSCSR RFYQLTKLLD SVQPIARELHQFTFDLLIKS HMVSVDFPEM MARIISVQVP KILSGKVKPI YFHTQ, as set forth inthree-letter abbreviation in SEQ ID NO:2.


2. A DNA expression vector for expressing a Macaca mulatta AR protein ina recombinant host cell wherein said expression vector comprises a DNAmolecule of claim
 1. 3. A host cell which expresses a recombinant Macacamulatta AR protein wherein said host cell contains the DNA expressionvector of claim
 2. 4. A process for expressing a Macaca mulatta ARprotein in a recombinant host cell, comprising: (a) transfecting theexpression vector of claim 2 into a suitable host cell; and (b)culturing the host cells of step (a) under conditions which allowexpression of said the Macaca mulatta AR protein from said DNAexpression vector.
 5. A purified DNA molecule encoding a Macaca mulattaAR protein wherein said protein consists of the amino acid sequence asfollows: MEVQLGLGRV YPRPPSKTYR GAFQNLFQSV REVIQNPGPR HPEAASAAPPGASLQQQQQQ QQETSPRQQQ QQQQGEDGSP QAHRRGPTGY LVLDEEQQPS QPQSAPECHPERGCVPEPGA AVAAGKGLPQ QLPAPPDEDD SAAPSTLSLL GPTFPGLSSC SADLKDILSEASTMQLLQQQ QQEAVSEGSS SGRAREASGA PTSSRDNYLE GTSTISDSAK ELCKAVSVSMGLGVEALEHL SPGEQLRGDC MYAPVLGVPP AVRPTPCAPL AECKGSLLDD SAGKSTEDTAEYSPFKGGYT KGLEGESLGC SGSAAAGSSG TLELPSTLSL YKSGALDEAA AYQSRDYYNFPLALAGPPPP PPPPHPHARI KLENPLDYGS AWAAAAAQCR YGDLASLHGA GAAGPGSGSPSAAASSSWHT LFTAEEGQLY GPCGGGGGGG GGGGGGAGEA GAVAPYGYTR PPQGLAGQEGDFTAPDVWYP GGMVSRVPYP SPTCVKSEMG PWMDSYSGPY GDMRLETAPD HVLPIDYYFPPQKTCLICGD EASGCHYGAL TCGSCKVFFK RAAEGKQKYL CASRNDCTID KFRRKNCPSCRLRKCYEAGM TLGARKLKKL GNLKLQEEGE ASSTTSPTEE TAQKLTVSHI EGYECQPIFLNVLEAIEPGV VCAGHDNNQP DSFAALLSSL NELGERQLVH VVKWAKALPG FRNLHVDDQMAVIQYSWMGL MVFAMGWRSF TNVNSRMLYF APDLVFNEYR MHKSRMYSQC VRMRHLSQEFGWLQITPQEF LCMKALLLFS IIPVDGLKNQ KFFDELRMNY IKELDRIIAC KRKNPTSCSRRFYQLTKLLD SVQPIARELH QFTFDLLIKS HMVSVDFPEM MAEIISVQVP KILSGKVKPI YFHTQ,as set forth in three-letter abbreviation in SEQ ID NO:2.


6. A DNA expression vector for expressing a Macaca mulatta AR protein ina recombinant host cell wherein said expression vector comprises a DNAmolecule of claim
 5. 7. A host cell which expresses a recombinant Macacamulatta AR protein wherein said host cell contains the expression vectorof claim
 6. 8. A process for expressing a Macaca mulatta AR protein in arecombinant host cell, comprising: (a) transfecting the expressionvector of claim 6 into a suitable host cell; and (b) culturing the hostcells of step (a) under conditions which allow expression of said theMacaca mulatta AR protein from said expression vector.
 9. A purified DNAmolecule encoding a Macaca mulatta AR protein wherein said DNA moleculecomprises the nucleotide sequence, as follows: CCCAAAAAAT AAAAACAAACAAAAACAAAA CAAAACAAAA AAAACGAATA AAGAAAAAGG TAATAACTCA GTTCTTATTTGCACCTACTT CCAGTGGACA CTGAATTTGG AAGGTGGAGG ATTCTTGTTT TTTCTTTTAAGATCGGGCAT CTTTTGAATC TACCCCTCAA GTGTTAAGAG ACAGACTGTG AGCCTAGCAGGGCAGATCTT GTCCACCGTG TCTCTTCTTT TGCAGGAGAC TTTGAGGCTG TCAGAGCGCTTTTTGCGTGG TTGCTCCCGC AAGTTTCCTT CTCTGGAGCT TCCCGCAGGT GGGCAGCTAGCTGCAGCGAC TACCGCATCA TCACAGCCTG TTGAACTCTT CTGAGCAAGA GAAGGGGAGGCGGGGTAAGG GAAGTAGGTG GAAGATTCAG CCAAGCTCAA GGATGGACGT GCAGTTAGGGCTGGGGAGGG TCTACCCTCG GCCGCCGTCC AAGACCTACC GAGGAGCTTT CCAGAATCTGTTCCAGAGCG TGCGCGAAGT GATCCAGAAC CCGGGCCCCA GGCACCCAGA GGCCGCGAGCGCAGCACCTC CCGGCGCCAG TTTGCAGCAG CAGCAGCAGC AGCAGCAAGA AACTAGCCCCCGGCAACAGC AGCAGCAGCA GCAGGGTGAG GATGGTTCTC CCCAAGCCCA TCGTAGAGGCCCCACAGGCT ACCTGGTCCT GGATGAGGAA CAGCAGCCTT CACAGCCTCA GTCAGCCCCGGAGTGCCACC CCGAGAGAGG TTGCGTCCCA GAGCCTGGAG CCGCCGTGGC CGCCGGCAAGGGGCTGCCGC AGCAGCTGCC AGCACCTCCG GACGAGGATG ACTCAGCTGC CCCATCCACGTTGTCTCTGC TGGGCCCCAC TTTCCCCGGC TTAAGCAGCT GCTCCGCCGA CCTTAAAGACATCCTGAGCG AGGCCAGCAC CATGCAACTC CTTCAGCAAC AGCAGCAGGA AGCAGTATCCGAAGGCAGCA GCAGCGGGAG AGCGAGGGAG GCCTCGGGGG CTCCCACTTC CTCCAAGGACAATTACTTAG AGGGCACTTC GACCATTTCT GACAGCGCCA AGCAGCTGTG TAAGGCAGTGTCGGTGTCCA TGGGCTTGGG TGTGGAGGCG TTGGAGCATC TGAGTCCAGG GGAACAGCTTCGGGGGGATT GCATGTACGC CCCAGTTTTG GGAGTTCCAC CCGCTGTGCG TCCCACTCCGTGTGCCCCAT TGGCCGAATG CAAAGGTTCT CTGCTAGACG ACAGCGCAGG CAAGAGCACTGAAGATACTG CTGAGTATTC CCCTTTCAAG GGAGGTTACA CCAAAGGGCT AGAAGGCGAGAGCCTAGGCT GCTCTGGCAG CGCTGCAGCA GGGAGCTCCG GGACACTTGA ACTGCCGTCCACCCTGTCTC TCTACAAGTC CGGAGCACTG GACGAGGCAG CTGCGTACCA GAGTCGCGACTACTACAACT TTCCACTGGC TCTGGCCGGG CCGCCGCCCC CTCCACCGCC TCCCCATCCCCACGCTCGCA TCAAGCTGGA GAACCCGCTG GACTATGGCA GCGCCTGGGC GGCTGCGGCGGCGCAGTGCC GCTATGGGGA CCTGGCGAGC CTGCATGGCG CGGGTGCAGC GGGACCCGGCTCTGGGTCAC CCTCAGCGGC CGCTTCCTCA TCCTGGCACA CTCTCTTCAC AGCCGAAGAAGGCCAGTTGT ATGGACCGTG TGGTGGTGGG GGCGGCGGCG GTGGCGGCGG CGGCGGCGGCGCAGGCGAGG CGGGAGCTGT AGCCCCCTAC GGCTACACTC GGCCACCTCA GGGGCTGGCGGGCCAGGAAG GCGACTTCAC CGCACCTGAT GTGTGGTACC CTGGCGGCAT GGTGAGCAGAGTGCCCTATC CCAGTCCCAC TTGTGTCAAA AGCGAGATGG GCCCCTGGAT GGATAGCTACTCCGGACCTT ACGGGGACAT GCGTTTGGAG ACTGCCAGGG ACCATGTTTT GCCAATTGACTATTACTTTC CACCCCAGAA GACCTGCCTG ATCTGTGGAG ATGAAGCTTC TGGGTGTCACTATGGAGCTC TCACATGTGG AAGCTGCAAG GTCTTCTTCA AAAGAGCCGC TGAAGGGAAACAGAAGTACC TGTGTGCCAG CAGAAATGAT TGCACTATTG ATAAATTCCG AAGGAAAAATTGTCCATCTT GCCGTCTTCG GAAATGTTAT GAAGCAGGGA TGACTCTGGG AGCCCGGAAGCTGAAGAAAC TTGGTAATCT GAAACTACAG GAGGAAGGAG AGGCTTCCAG CACCACCAGCCCCACTGAGG AGACAGGCCA GAAGCTGACA GTGTCACACA TTGAAGGCTA TGAATGTCAGCCCATCTTTC TGAATGTCCT GGAGGCCATT GAGCCAGGTG TGGTGTGTGC TGGACATGACAACAACCAGC CCGACTCCTT CGCAGCCTTG CTCTCTAGCC TCAATGAACT GGGAGAGAGACAGCTTGTAC ATGTGGTCAA GTGGGCCAAG GCCTTGCCTG GCTTCCGCAA CTTACACGTGGACGACCAGA TGGCTGTCAT TCAGTACTCC TGGATGGGGC TCATGGTGTT TGCCATGGGCTGGCGATCCT TCACCAATGT CAACTCCAGG ATGCTCTACT TTGCCCCTGA TCTGGTTTTCAATGAGTACC GCATGCACAA ATCCCGGATG TACAGCCAGT GTGTCCGAAT GAGGCACCTCTCTCAAGAGT TTGGATGGCT CCAAATCACC CCCCAGGAAT TCCTGTGCAT GAAAGCGCTGCTACTCTTCA GCATTATTCC AGTGGATGGG CTGAAAAATC AAAAATTCTT TGATGAACTTCGAATGAACT ACATCAAGGA ACTCGATCGT ATCATTGCAT GCAAAAGAAA AAATCCCACATCCTGCTCAA GGCGTTTCTA CCAGCTCACC AAGCTCCTGG ACTCCGTGCA GCCTATTGCGAGAGAGCTGC ATCAGTTCAC TTTTGACCTG CTAATCAAGT CACACATGGT GAGCGTGGACTTTCCGGAAA TGATGGCAGA GATCATCTCT GTGCAAGTGC CCAAGATCCT TTCTGGGAAAGTCAAGCCCA TCTATTTCCA CACCCAGTGA AGCATTGGAA ATCCCTATTT CCTCACCCCAGCTCATGCCC CCTTTCAGAT GTCTTCTGCC TGTTA, set forth as SEQ ID NO:1.


10. A DNA molecule of claim 9 which consists of nucleotide 154 to aboutnucleotide 1257 of SEQ ID NO:
 1. 11. An expression vector for expressinga Macaca mulatta AR protein wherein said expression vector comprises aDNA molecule of claim
 9. 12. An expression vector for expressing aMacaca mulatta AR protein wherein said expression vector comprises a DNAmolecule of claim
 10. 13. A host cell which expresses a recombinantMacaca mulatta AR protein wherein said host cell contains the expressionvector of claim
 11. 14. A host cell which expresses a recombinant Macacamulatta AR protein wherein said host cell contains the expression vectorof claim
 12. 15. A process for expressing a Macaca mulatta AR protein ina recombinant host cell, comprising: (a) transfecting the expressionvector of claim 11 into a suitable host cell; and, (b) culturing thehost cells of step (a) under conditions which allow expression of saidthe Macaca mulatta AR protein from said expression vector.
 16. Theprocess of claim 15 wherein the host cell is a yeast host cell.
 17. Apurified DNA molecule encoding a Macaca mulatta AR protein wherein saidDNA molecule consists of the nucleotide sequence, as follows, CCCAAAAAATAAAAACAAAC AAAAACAAAA CAAAACAAAA AAAACGAATA AAGAAAAAGG TAATAACTCAGTTCTTATTT GCACCTACTT CCAGTGGACA CTGAATTTGG AAGGTGGAGG ATTCTTGTTTTTTCTTTTAA GATCGGGCAT CTTTTGAATC TACCCCTCAA GTGTTAAGAG ACAGACTGTGAGCCTAGCAG GGCAGATCTT GTCCACCGTG TGTCTTCTTT TGCAGGAGAC TTTGAGGCTGTCAGAGCGCT TTTTGCGTGG TTGCTCCCGC AAGTTTCCTT CTCTGGAGCT TCCCGCAGGTGGGCAGCTAG CTGCAGCGAC TACCGCATCA TCACAGCCTG TTGAACTCTT CTGAGCAAGAGAAGGGGAGG CGGGGTAAGG GAAGTAGGTG GAAGATTCAG CCAAGCTCAA GGATGGAGGTGCAGTTAGGG CTGGGGAGGG TCTACCCTCG GCCGCCGTCC AAGACCTACC GACGAGCTTTCCAGAATCTG TTCCAGAGCG TGCGCGAAGT GATCCAGAAC CCGGGCCCCA GGCACCCAGAGGCCGCGAGC GCAGCACCTC CCGGCGCCAG TTTGCAGCAG CAGCAGCAGC AGCAGCAAGAAACTAGCCCC CGGCAACAGC AGCAGCAGCA GCAGGGTGAG GATGGTTCTC CCCAAGCCCATCGTAGAGGC CCCACAGGCT ACCTGGTCCT GGATGAGGAA CAGCAGCCTT CACAGCCTCAGTCAGCCCCG GAGTGCCACC CCGAGAGAGG TTGCGTCCCA GAGCCTGGAG CCGCCGTGGCCGCCGGCAAG GGGCTGCCGC AGCAGCTGCC AGCACCTCCG GACGAGGATG ACTCAGCTGCCCCATCCACG TTGTCTCTGC TGGGCCCCAC TTTCCCCGGC TTAAGCAGCT GCTCCGCCGACCTTAAAGAC ATCCTGAGCG AGGCCAGCAC CATGCAACTC CTTCAGCAAC AGCAGCAGGAAGCAGTATCC GAAGGCAGCA GCAGCGGGAG AGCGAGGGAG GCCTCGGGGG CTCCCACTTCCTCCAAGGAC AATTACTTAG AGGGCACTTC GACCATTTCT GACAGCGCCA AGGAGCTGTGTAAGGCAGTG TCGGTGTCCA TGGGCTTGGG TGTGGAGGCG TTGGAGCATC TGAGTCCAGGGGAACAGCTT CGGGGGGATT GCATGTACGC CCCAGTTTTG GGAGTTCCAC CCGCTGTGCGTCCCACTCCG TGTGCCCCAT TGGCCGAATG CAAAGGTTCT CTGCTAGACG ACAGCGCAGGCAAGAGCACT GAAGATACTG CTGAGTATTC CCCTTTCAAG GGAGGTTACA CCAAAGGGCTAGAAGGCGAG AGCCTAGGCT GCTCTGGCAG CGCTGCAGCA GGGAGCTCCG GGACACTTGAACTGCCGTCC ACCCTGTCTC TCTACAAGTC CGGAGCACTG GACGAGGCAG CTGCGTACCAGAGTCGCGAC TACTACAACT TTCCACTGGC TCTGGCCGGG CCGCCGCCCC CTCCACCGCCTCCCCATCCC CACGCTCGCC TCAAGCTGGA GAACCCGCTG GACTATGGCA GCGCCTGGGCGGCTGCGGCG GCGCAGTGCC GCTATGGGGA CCTGGCGAGC CTGCATGGCG CGGGTGCAGCGGGACCCGGC TCTGGGTCAC CCTCAGCGGC CGCTTCCTCA TCCTGGCACA CTCTCTTCACAGCCGAAGAA GGCCAGTTGT ATGGACCGTG TGGTGGTGGG GGCGGCGGCG GTGGCGGCGGCGGCGGCGGC GCAGGCGAGG CGGGAGCTGT AGCCCCCTAC GGCTACACTC GGCCACCTCAGGGGCTGGCG GGCCAGGAAG GCGACTTCAC CGCACCTGAT GTGTGGTACC CTGGCGGCATGGTGAGCAGA GTGCCCTATC CCAGTCCCAC TTGTGTCAAA AGCGAGATGG GCCCCTGGATGGATAGCTAC TCCGGACCTT ACGGGGACAT GCGTTTGGAG ACTGCCAGGG ACCATGTTTTGCCAATTGAC TATTACTTTC CACCCCAGAA GACCTGCCTG ATCTGTGGAG ATGAAGCTTCTGGGTGTCAC TATGGAGCTC TCACATGTGG AAGCTGCAAG GTCTTCTTCA AAAGAGCCGCTGAAGGGAAA CAGAAGTACC TGTGTGCCAG CAGAAATGAT TGCACTATTG ATAAATTCCGAAGGAAAAAT TGTCCATCTT GCCGTCTTCG GAAATGTTAT GAAGCAGGGA TGACTCTGGGAGCCCGGAAG CTGAAGAAAC TTGGTAATCT GAAACTACAG GAGGAAGGAG AGGCTTCCAGCACCACCAGC CCCACTGAGG AGACAGCCCA GAAGCTGACA GTGTCACACA TTGAAGGCTATGAATGTCAG CCCATCTTTC TGAATGTCCT GGAGGCCATT GAGCCAGGTG TGGTGTGTGCTGGACATGAC AACAACCAGC CCGACTCCTT CGCAGCCTTG CTCTCTAGCC TCAATGAACTGGGAGAGAGA CAGCTTGTAC ATGTGGTCAA GTGGGCCAAG GCCTTGCCTG GCTTCCGCAACTTACACGTG GACGACCAGA TGGCTGTCAT TCAGTACTCC TGGATGGGGC TCATGGTGTTTGCCATGGGC TGGCGATCCT TCACCAATGT CAACTCCAGG ATGCTCTACT TTGCCCCTGATCTGGTTTTC AATGAGTACC GCATGCACAA ATCCCGGATG TACAGCCAGT GTGTCCGAATGAGGCACCTC TCTCAAGAGT TTGGATGGCT CCAAATCACC CCCCACGAAT TCCTGTGCATGAAAGCGCTG CTACTCTTCA GCATTATTCC AGTGGATGGG CTGAAAAATC AAAAATTCTTTGATGAACTT CGAATGAACT ACATCAAGGA ACTCGATCGT ATCATTGCAT GCAAAAGAAAAAATCCCACA TCCTGCTCAA GGCGTTTCTA CCAGCTCACC AAGCTCCTGG ACTCCGTGCAGCCTATTGCG AGAGAGCTGC ATCAGTTCAC TTTTGACCTG CTAATCAAGT CACACATGGTGAGCGTGGAC TTTCCGGAAA TGATGGCAGA GATCATCTCT GTGCAAGTGC CCAAGATCCTTTCTGGGAAA GTCAAGCCCA TCTATTTCCA CACCCAGTGA AGCATTGGAA ATCCCTATTTCCTCACCCCA GCTCATGCCC CCTTTCAGAT GTCTTCTGCC TGTTA, as set forth in SEQID NO:
 1.


18. A DNA molecule of claim 17 which consists of nucleotide 423 to aboutnucleotide 3108 of SEQ ID NO:
 1. 19. A DNA expression vector forexpressing a Macaca mulatta AR protein wherein said expression vectorcomprises a DNA molecule of claim
 17. 20. A DNA expression vector forexpressing a Macaca mulatta AR protein wherein said expression vectorcomprises a DNA molecule of claim
 18. 21. A host cell which expresses arecombinant Macaca mulatta AR protein wherein said host cell containsthe expression vector of claim
 19. 22. A host cell which expresses arecombinant Macaca mulatta AR protein wherein said host cell containsthe expression vector of claim
 20. 23. A process for expressing a Macacamulatta AR protein in a recombinant host cell, comprising: (a)transfecting the expression vector of claim 19 into a suitable hostcell; and (b) culturing the host cells of step (a) under conditionswhich allow expression of said the Macaca mulatta AR protein from saidexpression vector.
 24. The process of claim 23 wherein the host cell isa yeast host cell.